Glycobiology of Human Milk Oligosaccharides

Transcription

www.glycom.com
The First International Conference on the
May 16 & 17, 2011
Tivoli Hotel, Copenhagen, Denmark
BOOK OF ABSTRACTS
www.glycom.com
The First International Conference on the
May 16 & 17, 2011
Tivoli Hotel, Copenhagen, Denmark
Main topics
Significance of Carbohydrates
HMO Metabolism in Humans
Microbial Colonization and HMOs metabolism by microbiota
HMO Content & Composition: New Analytical Approaches
Potential of HMOs & Principal Components in Health and Disease
Organizing Committee
Professor Clemens Kunz, University of Giessen, Germany
Professor Sharon Donovan, University of Illinois, USA
Christoph Röhrig, Judit Kovacs & Susanne Mau, Glycom A/S, Denmark
Website: www.glycom.com
CONTENTS
Contents
P 01
Contents
P 03
Conference program
P 05
Conference dinner
Abstracts of plenary lectures and oral communications
P 07
CLEMENS KUNZ (Germany): Research on HMOs – A brief historical review
P 08
HUDSON FREEZE (USA): Sweet solution: Sugars for health?
P 09
PEDRO ANTONIO PRIETO et al. (Mexico): Human Milk Oligosaccharides: from synthesis to clinical tests and collateral
observations
P 10
TADASU URASHIMA et al. (Japan): Possible significance of the predominance of type 1 oligosaccharides, a human specific
feature, in breast milk
P 11
CLEMENS KUNZ et al. (Germany): In vivo 13C-labeling of milk oligosaccharides and their metabolism in infants
P 12
SHARON M. DONOVAN et al. (USA): Transcriptome of the human infant intestinal ecosystem
P 13
THIERRY HENNET et al. (Switzerland): The role of milk sialyllactose on intestinal bacterial colonization
P 14
DAVID A. MILLS (USA): Nursing our microbiota: Interactions between bifidobacteria and milk oligosaccharides
P 15
MOTOMITSU KITAOKA (Japan): Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides
P 16
DAVID S. NEWBURG et al. (USA): Human milk oligosaccharides alter human faecal microbiota during in vitro
fermentation
P 17
DANIEL GARRIDO et al. (USA): Characterization of glycosyl hydrolases in bifidobacterium longum subsp. infantis active
on human milk oligosaccharides
P 18
DEVON KAVANAUGH et al. (Ireland): Increased adherence of bifidobacterium longum subsp. infantis to HT-29 cells
following exposure to a predominant human milk oligosaccharide
P 19
RUDOLF GEYER et al. (Germany): Strategies for screening and structural characterization of HMOs
P 20
CARLITO B. LEBRILLA (USA): High throughput analysis and quantitation of free oligosaccharides
P 21
JOHN S. KLASSEN et al. (Canada): Quantifying human milk oligosaccharide-protein interactions in vitro using
electrospray ionization mass spectrometry
P 22
DENNIS BLANK et al. (Germany): Lewis blood group detection from human milk oligosaccharide mass finger prints
P 23
SHUAI WU et al. (USA): The development of an annotated structure library for human milk oligosaccharides
P 24
UTE KRENGEL et al. (Norway): Blood group dependence of cholera infections
P 25
NORBERT SPRENGER (Switzerland): Sialic acid utilization
P 26
BING WANG (China): Nutritional significance of sialic acid in human milk: an essential nutrient for brain development
and cognition
P 27
LARS BODE et al. (USA): Human milk oligosaccharides in amebiasis and necrotizing enterocolitis
P 28
SØRGE KELM et al. (Germany): Interactions of milk glycoconjugates with siglecs
P 29
JASMINE GRINYER et al. (Australia): Protein-sugar interactions between secreted fluids and pathogens as a protective
mechanism
P 31
Short biographies of invited speakers
1
CONTENTS
Contents
Abstracts of posters
P 35
WAI YUEN CHEAH et al. (Australia): Role of sialic acid in innate immune protection provided by mammalian milk
P 36
SHARON M. DONOVAN et al. (USA): Ascending colonic microbiota composition and SCFA patterns produced from in
vitro fermentation of human milk oligosaccharides and prebiotics differ between formula-fed and sow-reared
piglets
P 37
VICTORIA DOTZ et al. (Germany): Oligosaccharide MALDI-MS profiles in milk and urine from mother-child pairs
P 38
AMR EL-HAWEIT et al. (Canada): Human milk oligosaccharides as anti-adhesion candidates for clostridium difficile
toxin
P 39
SABRINA ETZOLD et al. (UK): Structure determination of bacterial mucus-binding proteins and their functional role
in adhesion to host glycans
P 40
LEONIDES FERNÁNDEZ et al. (Spain): Breast milk microbiota: is there a relationship with HMOs?
P 41
SURI S. IYER (USA): Tailoring carbohydrates to capture toxins and pathogens
P 42
TAKANE KATAYAMA et al. (Japan): α-L-Fucosynthase that specifically introduces Lewis a/x antigens into type-1/2
chains
P 43
JONATHAN A. LANE et al. (Ireland): A new methodology for screening of bacteria-carbohydrate interactions: antiadhesive milk oligosaccharides as a case study
P 44
MAGDALENA ORCZYK-PAWIŁOWICZ et al. (Poland): Sialylation and fucosylation of human milk α1-acid glycoprotein
during the first two weeks of lactation
P 45
MAGDALENA ORCZYK-PAWIŁOWICZ et al. (Poland): The relative amounts of fucose isoforms in oligosaccharides of
human milk fibronectin
P 46
KRISTINE A. SCHOLAND et al. (Germany): Effects of specific milk oligosaccharides on the expression of interleukin-8
and marker enzymes of intestinal cell maturation
P 47
KATELYN ZAK et al. (USA): In vivo production of fucose-α1, 2-lactose
P 48
BETSY YANG et al. (USA & Taiwan): Sialylated galactosides of human milk as inhibitors of enterovirus 71 and A
(H1N1) 2009 influenza infections
P 49
ARDYTHE L. MORROW et al. (USA): The oligosaccharide (OS) phenotype of preterm infants predicts risk: A potential
indication for HMOS administration?
P 50
MAKSIM NAVAKOUSKI et al. (Russia): Natural antibodies against milk oligosaccharides
P 51
GER T. RIJKERS et al. (The Netherlands): Primary prevention of allergic diseases by probiotics: impact of HMOs
P 52
The Sponsor: Glycom
P 53
List of participants
2
CONFERENCE PROGRAM
Conference Program
Monday, May 16, 2011
08.00
Registration & Coffee
09.00
Welcome by John Theroux, Glycom & Clemens Kunz, University of Giessen, GERMANY
Chairs: Sharon Donovan, Bing Wang
09.15
09.50
10.25
10.50
Research on HMOs – A brief historical review
Clemens Kunz, Ph.D. – University of Giessen, GERMANY
Sweet Solution: Sugars for Health?
Hudson Freeze, Ph.D. – Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
Human Milk Oligosaccharides: From Synthesis to Clinical Tests and Collateral Observations
Pedro Antonio Prieto, Ph.D. – Tecnológico de Monterrey, Health Sciences Division, Monterrey, MEXICO
Break and Poster Viewing & Coffee
Chairs: David Mills, Norbert Sprenger
11.15
11.50
12.25
13.00
Type 1 HMO Predominance: A Specific Feature in Human Milk
Tadasu Urashima, Ph.D. – Obihiro University, Hokkaido, JAPAN
In vivo 13C-labeling of Milk Oligosaccharides and Metabolism in Infants
Transcriptome of the Human Infant Intestinal Ecosystem
Sharon Donovan Ph.D., R.D. – University of Illinois, Urbana, USA
Lunch
Microbial Colonization and HMOs Metabolism by Microbiota
Chairs: Hudson Freeze, Carlito Lebrilla
14.00
14.35
15.10
15.30
16. 05
16.30
The Role of Milk Sialyllactose on Intestinal Bacterial Colonization
Thierry Hennet, Ph.D. – University of Zurich, SWITZERLAND
Nursing Our Microbiota: Interactions Between Bifidobacteria and Milk Oligosaccharides
David Mills, Ph.D. – University of California, Davis, USA
Bifidobacterial Enzymes Involved in the Metabolism of Human Milk Oligosaccharides
Motomitsu Kitaoka, Ph.D. – National Food Research Institute, Ibaraki, JAPAN
Human Milk Oligosaccharides alter Human Fecal Microbiota During In Vitro Fermentation
David S. Newburg, Ph.D. – Boston College, Chestnut Hill, MA, USA
Characterization of Glycosyl Hydrolases in Bifidobacterium longum subsp. infantis active on Human
Milk Oligosaccharides
Daniel Garrido – University of California, Davis, USA
16.45
Increased Adherence of Bifidobacterium longum subsp. infantis to HT-29 cells following Exposure to a
Predominant Human Milk Oligosaccharide
Devon Kavanaugh – Teagasc Food Research Centre, Cork, National University of Ireland, IRELAND
17.00
END OF SESSION
18.00
Meet in Lobby – Leave for Tivoli Gardens at 18.15
19.00
Dinner at Tivoli Gardens (Reception opens at 18.30)
3
CONFERENCE PROGRAM
Conference Program
Tuesday, May 17, 2011
Chairs: Tadasu Urashima, Lars Bode
08.30
09.05
09.40
Strategies for Screening and Structural Characterization of HMOs
Rudolf Geyer, Ph.D. – University of Giessen, GERMANY
High Throughput Analysis and Quantitation of Free Oligosaccharides in Mammalian Milk
Carlito Lebrilla, Ph.D. – University of California, Davis, USA
Quantifying Human Milk Oligosaccharide-Protein Interactions In Vitro using Electrospray Ionization
Mass Spectrometry
John Klassen, Ph.D. – University of Alberta, CANADA
10.05
10.25
10.40
Lewis Blood Group Detection from Human Milk Oligosaccharide Mass Finger Prints
Dennis Blank – University of Giessen, GERMANY
The Development of an Annotated Structure Library for Human Milk Oligosaccharides
Shuai Wu – University of California, Davis, USA
10.55
Blood Group Dependence of Cholera Infections
11.10
Poster Session & Coffee
13.00
Lunch
Ute Krengel, Ph.D. – University of Oslo, NORWAY
Chairs: Rudolf Geyer, Thierry Hennet
14.00
14.35
15.10
15.20
15.55
16.20
Sialic Acid Utilization
Norbert Sprenger, Ph.D. – Nestlé Research Center, Lausanne, SWITZERLAND
The Nutritional Significance of Sialic Acid on Neurodevelopment and Cognition
Bing Wang, Ph.D. – University of Sydney, Australia; Xiamen University & Nestlé Research Center, Beijing, P. R. CHINA
Coffee break
Human Milk Oligosaccharides in Amebiasis and Necrotizing Enterocolitis
Lars Bode, Ph.D. – University of California, San Diego, USA
Interactions of Milk Glycoconjugates with Siglecs
Sørge Kelm, PhD. – University Bremen, GERMANY
Protein-Sugar Interactions between Secreted Fluids and Pathogens as a Protective Mechanism
Jasmine Grinyer – Macquarie University, Sydney NSW, AUSTRALIA
Summary of Conference
16.35
Sharon Donovan Ph.D., R.D. – University of Illinois, Urbana, USA
4
CONFERENCE DINNER
Conference Dinner at Tivoli Gardens
The Reception for the Conference Dinner starts on Monday evening at 18.30 in the H. C. Andersen
Castle inside Tivoli Gardens (① on the map). The Dinner will start at 19.00.
To enter Tivoli Gardens you will require the entrance & dinner ticket that you received together with
your conference material if you signed up for the dinner in time.
If you stay at the Tivoli Hotel we recommend that you come to the Lobby at 18.00. To Tivoli Gardens it
is approximately a 10 minute walk from the Hotel.
If you stay at another location, please use the map to find Tivoli Gardens and ask then at the entrance
for the H. C. Andersen Castle (Tivoli Gardens is big and there are several restaurants).
5
CONFERENCE DINNER
Conference Dinner at Tivoli Gardens
Menu
Roasted scallop with lamb’s lettuce and pesto
(Ristet kammusling med vårsalat og pesto)
Fillet of beef with mild rose pepper, pancetta and beans
(Oksefilet med mild rosenpeber, pancetta og bønner)
Chocolate cake Marie José with preserved rhubarb and créme Anglaise
(Chokoladekage Marie José med syltede rabarber og creme anglaise)
Wines
Casa Mayor Chile, Chardonnay and Merlot
6
ABSTRACTS OF PLENARY LECTURES & ORAL COMMUNICATIONS
Abstracts of Plenary Lectures & Oral Communications
Research on HMOs – A brief historical review
CLEMENS KUNZ
Institute of Nutritional Science, University of Giessen, Germany
The early history of human milk oligosaccharides (HMOs) comprises research focusing on chemical milk
composition in general, but also on physiological and nutritional aspects and chemical structure determinations.
At the end of the 19th century, human milk and cow’s milk were thought to contain a mixture of lactoses
differing strongly in their properties. Between the 1920s and 1950s, it was found that this lactose fraction was
composed of a mixture of components called “gynolactose”. Their essential feature was the presence of nitrogen
and hexosamines. Pediatricians observed that in feces of breast-fed infants compared to infants receiving
formula, bifidobacteria were the predominant microorganisms; oligosaccharide fractions containing Nacetylglucosamine were growth promoting and thus named “bifidus factor”. At the same time, the structures of
major HMOs such as lacto-N-tetraose, neo-lacto-N-tetraose and their fucosylated derivatives, as well as sialylated
and fucosylated lactose have been identified. The last 30 years have been dominated by the development of
powerful analytical tools to identify and characterize HMOs and by a vast number of functional studies in vitro.
Today, due to the enormous biotechnological progress we are at the beginning of a new era focusing on specific
effects of HMOs in animals and humans.
7
Sweet solution: Sugars for health?
HUDSON FREEZE
Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
There’s little surprise when sugars, energy and life appear in the same sentence, but sugars are much more than nutrition and
energy. Complex carbohydrates, polysaccharides, glycans, call them what you will, are seldom well known and their
physiological functions are usually underappreciated. Glycan synthesis begins with activation of selected monosaccharides
and their localization next to biosynthetic enzymes for assembly, remodeling and recycling in the glycan biosphere. The flat
metabolic chart inadequately reflects the complexity of the cellular organization, regulation and balance inherent in these
competing and complimentary homeostatic processes.
The shear complexity of grappling with human health drives medicine to define and conceptualize organ systems and
enumerate their pathogenic symptoms. But the fundamental rules of basic science respect no artificial constructs. Combine
that fact with an under appreciation of the diversity of glycan structure and function, and it leaves the current medical system
ill-prepared to deal with glycan based pathologies arising from genetic or environmental insults. Here are a few examples of
the consequences and complexities of faulty monosaccharide biosynthetic pathways.
Congenital disorder of glycosylation (CDG) Type Ib (CDG-Ib) patients show failure to thrive, numerous gastrointestinal
problems, hypoglycemia, and liver fibrosis due to a phosphomannose isomerase deficiency (MPI, Fruc-6-P→ Man-6-P).
Providing oral mannose supplements reverse all symptoms except fibrosis because mannose bypasses the deficient step.
Good. In mice, complete loss of MPI is embryonic lethal and additional mannose only hastens their demise due to Man-6-P
accumulation and ATP depletion. Not good. Make a hypomorphic mouse line with patient-levels of residual MPI activity
(15%) and embryos survive, but adult mice reach old age showing few if any pathologies; obviously not a model for CDG-Ib.
But stress young mice on a high fat diet, and they gain far less weight than their wild-type brothers. That seems a good
outcome. However, give a pregnant hypomorphic mouse mannose supplements in her drinking water and she aborts all the
hypomorphic progeny. Reduce the amount of mannose and embryos survive, but half of the progeny are blind by 5 weeks of
age, because of abnormal eye development during gestation. So mannose is good, bad, and ugly in different scenarios. What
does this mean for MPI-deficient patients, heterozygous parents or individuals using mannose as a therapy or prevention of
E. coli-based urinary tract infections? The answer is unknown, but cautious use of this simple sugar may be wise.
Some patients who have a fucosylation deficiency due to the loss of a GDP-Fucose transporter in the Golgi normalize
their elevated circulating neutrophils within a few days of being provided fucose. Other patients with mutations in the same
gene are unresponsive. Experiments in mouse models of this disorder show that controlling leukocyte number depends on
appropriate fucosylation of proteins in the critical Notch signaling pathway.
Supplements of N-acetylglucosamine were reported to reverse serious intestinal pathology some among children with
Crohn’s disease. These findings led to testing oral N-acetylglucosamine as a treatment for T-cell mediated autoimmunity in a
mouse model with good results.
Recently, a deficiency in the ER-localized glucose-6-P’ase3 (G6PC3) was found to cause ER stress and compromise
glycosylation of gp91 component of the NADPH oxidase, resulting in neutropenia. Moreover, patients with glycogen storage
disorder (GSD) 1b due to a glucose-6-P translocase deficiency show similar neutrophil dysfunction. These results suggest that
glucose homeostasis will be important in not only nutrition, but also in the biosynthesis of both N- and O-glycans.
These examples show that manipulating the metabolic flux of monosaccharides can have marked influences on protein
glycosylation and health in different organ systems. Basic glycobiology underlies and interconnects multiple medical
disciplines for the benefit of the most important stakeholders, the patients and their families.
Supported by The Rocket Fund and a Sanford Professorship in Glycobiology
8
Human Milk Oligosaccharides From Synthesis to Clinical Tests and Collateral Observations
PEDRO ANTONIO PRIETO1 AND JAMES LEACH2
1Tecnológico
2Abbott
de Monterrey, Health Sciences Division, Monterrey, Mexico 66250
Nutrition, Columbus, Ohio, United States 43219
During the decade of the 90s and the first years of the present century, a group of scientists and engineers
sponsored by Abbott Laboratories embarked in the endeavor of synthesizing and testing human milk
oligosaccharides. By the year 2000 it was possible to synthesize and test kilogram amounts of the core
oligosaccharide Lacto-N-neotetraose which, in turn, allowed testing of this structure in clinical settings. In
parallel fashion, we explored the production of oligosaccharides in the milk of transgenic animals, in vitro
prebiotic activity of carbohydrates and an extensive analysis of human milk samples to ascertain their contents
of neutral oligosaccharide structures. Some of our findings emerged from fortuitous observations such as the
ability of dried yeast to drive oligosaccharide synthesis in the presence of suitable glycosyltransferases; others
were predicted from previously published accounts such as the variability of oligosaccharide content in human
milk samples. Yet other observations were unexpected and contradicted previously published data such as the
existence of oligosaccharide profiles devoid of fucose-containing structures and the ability of fucosylated
glycoconjugates to shot down milk production in transgenic rabbits. Taken together, the results that emerged
from this project demonstrate that there are no longer obstacles for the clinical testing of certain oligosaccharide
structures and that transgenic expression of secondary gene products in milk is possible in some species but not
in others. The present account summarizes the evolution of a project from its inception to the evaluation of
scientifically relevant facts that emerged as byproducts in the quest of attaining access to free soluble
carbohydrate structures from human milk.
Funded by: Abbott Nutrition, Tecnológico de Monterrey
9
Possible significance of the predominance of type 1 oligosaccharides, a human specific feature,
in breast milk
TADASU URASHIMA1, SADAKI ASAKUMA2, MICHAEL MESSER3, OLAV T. OFTEDAL4
1Obihiro
University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, Japan. 2National Agriculture Research
Center for Hokkaido Region, Sapporo, Japan. 3School of Molecular and Microbial Biosciences, The University of
Sydney, Australia. 4Smithsonian Environmental Research Center, Edgewater, USA.
Human milk and colostrum contain 12 ~ 13 g/L and 22 ~ 24 g/L of oligosaccharides, respectively. The chemical
structures of 115 human milk oligosaccharides (HMOs) have been characterized to date. We determined the
concentrations of 10 neutral and 9 acidic colostrum HMOs collected during the first 3 days of lactation, using
reverse phase HPLC after derivatization with 2-aminopyridine or 1-methyl-3-phenyl-5-pyrazolon.1,2 The
predominant oligosaccharides were found to be Fuc(α1-2)Gal(β1-4)Glc (2'-FL), Fuc(α1-2)Gal(β1-3)GlcNAc(β13)Gal(β1-4)Glc (LNFP1), Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc (LNDFH1) and Gal(β13)GlcNAc(β1-3)Gal(β1-4)Glc (LNT), the concentration of each of which was 1 ~ 3 g/L. As these HMOs, other than
2'-FL, all contain Gal(β1-3)GlcNAc (LNB, type 1), we conclude that HMOs containing the type 1 structure
predominate over other those containing Gal(β1-4)GlcNAc (LacNAc, type 2). This appears to be a feature that is
specific to humans since the milk/colostrum of other species, including apes and monkeys, either contain only
type 2 oligosaccharides or the type 2 predominate over the type 1.3,4,5
It was shown in another study that the relative concentration of LNT, when compared with other HMOs, is
significantly lower in the feces of breast fed infants than in breast milk, suggesting that LNT is metabolized in
preference to other HMOs within the infant colon.6 In addition, specific oligosaccharides such as Gal(β14)GlcNAc(β1-6)Gal(β1-4)Glc or Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)Gal(β1-4)Glc were found in these feces. These
type 2 oligosaccharides were presumably formed from LNH (Gal(β1-3)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β16)]Gal(β1-4)Glc) or monofucosyl LNH (Gal(β1-3)GlcNAc(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β14)Glc) by the release of LNB during the passage of HMOs through the infant colon, and were relatively resistant
to further degradation.
We hypothesize that LNB released from type 1 in preference to type 2 HMOs by Bifidobacterium bifidum
lacto-N-biosidase can be utilized by other strains of bifidobacteria within the colon of fed infants. The
predominance of type 1 HMOs may therefore promote the growth of beneficial colonic bifidobacteria, an effect
which may be stronger than that for type 2 HMOs. Thus acquisition of the predominance of these prebiotic type 1
HMOs may have had a selective advantage, in terms of survival of newborn human infants, during human
evolution.
1S.
Asakuma et al., Biosci. Biotechnol. Biochem. 71, 1447-1451, 2007.
2S.
Asakuma et al., Eur. J. Clin. Nutr. 62, 488-494, 2008.
3T.
Urashima et al., In: Comprehensive Glycoscience (J. P. Y. Kamerling ed.), pp. 695-724, Elservier, Amserdam, 2007.
4T.
Urashima et al., Glycobiology 19, 499-508, 2009.
5K.
Goto et al., Glycoconj. J. 27, 703-715, 2010.
6S.
Albrecht et al., Electrophoresis 31, 1264-1273, 2010.
10
In vivo 13C-labeling of milk oligosaccharides and their metabolism in infants
CLEMENS KUNZ1 AND SILVIA RUDLOFF2
1Institute
of Nutritional Science, University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany
2Department
of Pediatrics, University of Giessen, Feulgenstrasse 12, 35392 Giessen, Germany
There is increasing evidence that HMOs may be of particular importance for the infant. Functions which are
discussed include anti-adhesive and anti-inflammatory effects, an influence on brain development or preventive
effects with regard to certain diseases.
With regard to animal and human studies more knowledge on the metabolic fate of HMOs is needed. The use of
stable isotope (13C)-labelled HMOs facilitates such investigations as it allows the sensitive determination of the
13C-enrichment
in biological samples such as feces, blood or urine. The combination of isotope ratio mass
spectrometry and other mass spectrometric methods is well suited to answer metabolic questions in humans
and animals. In a previous study, we orally applied 13C-galactose to lactating mothers investigating whether or
not this monosaccharide was directly used for the biosynthesis of the large amounts of lactose (50-70 g/L) as
well as of oligosaccharides (5-15 g/L) in milk without being metabolized first-pass by the liver. We hypothesized
that it could be of advantage to offer galactose instead of glucose to the lactating mammary cell because (a) milk
biosynthesis can be turned on to a maximum within few minutes, (b) the amount of milk produced by a woman
can reach up to several liters a day and (c) galactose but not glucose is a main component of HMOs. As the
resulting in vivo- labelling of HMOs was very effective, we subsequently addressed metabolic questions in the
infant by analyzing the 13C-enrichment in faeces or in urine. The data obtained so far will be compared with the
current knowledge on metabolic aspects.
Interest in metabolic questions with regard to HMOs in infants started about 40 years ago when Lundblad
and co-workers in Lund (Sweden) and Strecker´s group in Lille (France) observed that renal excretion of
oligosaccharides in an infant was partly influenced by the oligosaccharide pattern of the mother’s milk. Today,
questions regarding HMOs which are of particular importance in the infant comprise (i) their potential
utilization by the gut microbiota, (ii) physiological processes within the whole digestive tract (from mouth to
colon), (iii) mechanisms of intestinal absorption of HMOs and (iv) their renal excretion.
Supported by the German Research Foundation (DFG Ru 529/7-3 and Ku 781/8-3).
11
Transcriptome of the Human Infant Intestinal Ecosystem
SHARON M. DONOVAN1, MEI WANG1, SHUAI WU2, CARLITO B. LEBRILLA2, SCOTT L. SCHWARTZ3,4, IVAN V. IVANOV3,4,
LAURIE A. DAVIDSON3, JENNIFER S. GOLDSBY3, DAVID B. DAHL5, EDWARD R. DOUGHERTY6, IDDO FRIEDBERG7,
DAMIR HERMAN8 AND ROBERT S. CHAPKIN2
1Department
of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801 USA. 2Department of
Chemistry, University of California, Davis, CA 95616 USA. 3Program in Integrative Nutrition, Center for
Environmental & Rural Health, 4Departments of Statistics, 5Veterinary Physiology & Pharmacology, and 6Electrical
Engineering, Texas A&M University, College Station, TX, 77843 USA. 7Departments of Microbiology and Computer
Science & Software Engineering, Miami University, Oxford, OH 45056 USA. 8Winthrop Rockefeller Cancer Institute,
University of Arkansas, Little Rock, AR 72204 USA
Our long-term goal is to use non-invasive approaches to define how early nutrition influences intestinal
development and shapes host-microbe interactions in the intestine of breast- (BF) and formula (FF) -fed infants.
Human milk contains a rich diversity of oligosaccharides (HMO) that serve as substrates for fermentation, act as
prebiotics for beneficial bacteria, block the attachment of pathogens and modulate immune development of the
infant. We have developed a novel molecular methodology that utilizes stool samples containing intact sloughed
epithelial cells to quantify intestinal gene expression profiles in the developing human neonate (AJP
2010;298:G582-9). Stool samples were collected from 3-month-old FF (n=10) and BF (n=12) infants and gene
expression was assessed by microarray analysis. Linear Discriminant Analysis (LDA) identified single genes and
the two- to three-gene combinations that distinguished the feeding groups. In addition, putative "master"
regulatory genes were identified using Coefficient of Determination analysis. Recently, this database was
extended to include breastmilk HMO composition determined by GC/MS, infant stool short chain fatty acids
(SCFA) by GC and infant microbiota composition and gene expression (in a subset of samples, n=6/group) by
Roche 454 metagenomic pyrosequencing. 2-Fucosyllactose (2’FL) was predominant in the milk of 3 mothers,
whereas lacto-N-tetraose (LNT) predominated in 3 mothers. Total SCFA and propionate concentrations were
greater in stool from FF vs. BF infants. Similar to host mRNA expression, bacterial DNA phylogenetic profiles
provided strong feature sets that clearly classified FF vs. BF babies. Several quantitative approaches for
integrating host cell gene expression and the bacterial phyla profiles were applied. First, LDA was used to predict
single gene expression for each of the 16,853 host genes using the percentage of Firmicutes and Actinobacteria
present in stool as covariates. For 394 genes, all model coefficients were significant (R 2 >0.7; q-values <0.25).
Next, correlations between the percentages of Carbohydrates, Virulence, Cell Wall-Capsule, and RNA metabolic
pathways in the microbiome (as categorized by SEED level 1 biological processes) and 519 host Immunity and
Defense genes (as categorized by PANTHER biological processes) were investigated. Strong correlations (R 2 >
0.7, q-values <0.2) were observed for 20 genes. Ongoing analyses involve a deeper examination of the
hierarchical SEED classifications relative to FF vs. BF. Our data suggest that a systems biology approach, such as
computational modeling, which integrates information from the breastmilk, infant and the microbiome can
identify important mechanistic pathways affecting intestinal development in the first few months of life.
Funded by: NIH R01 HD061929 , CA129444, and DNS Vision 20/20.
12
Microbial Colonization and HMOs Metabolism by Microbiota
The role of milk sialyllactose on intestinal bacterial colonization
ANDREA FUHRER1, NORBERT SPRENGER2, EKATERINA KURAKEVICH1, LUBOR BORSIG1, YEN-LIN HUANG1,
CHRISTOPHE CHASSARD3 AND THIERRY HENNET1
1Institute
2Nestlé
of Physiology and Center for Integrative Human Physiology, University of Zurich, Switzerland
Research Center, Vers-chez-les-Blanc, Lausanne, Switzerland
3Laboratory
of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
Milk oligosaccharides influence the composition of intestinal microbiota and thereby mucosal inflammation.
Recently, we have shown that mice fed on milk deficient for sialyl(2,3)lactose were more resistant to dextran
sulfate sodium (DSS)-induced colitis. By contrast, the exposure to milk containing or deficient for
sialyl(2,3)lactose had no impact on the development of mucosal leukocyte populations. The resistance to DSSinduced colitis was specifically related to sialyl(2,3)lactose since mice exposed to sialyl(2,6)lactose-deficient
milk did not react differently than mice exposed to normal milk. Considering the long term protection of
sialyl(2,3)lactose-deficient feeding, we have analysed and compared the composition of intestinal microbiota in
mice exposed to normal or sialyl(2,3)lactose-deficient milk during lactation. Using temperature-gradient gel
electrophoresis and real-time PCR, we did find that milk sialyl(2,3)lactose mainly affected the colonization of
the intestine by clostridial cluster IV bacteria. The resulting intestinal microbiota presented different metabolic
properties as shown by changes at the level of short-chain fatty acid production in the caecum of mice exposed to
either normal milk or sialyl(2,3)lactose-deficient milk. The relationship between intestinal microbiota and the
severity of DSS-induced colitis was demonstrated by reconstituting germ-free mice with intestinal microbiota
isolated from mice fed on normal milk or sialyl(2,3)lactose-deficient milk. Germ-free mice harbouring
microbiota from sialyl(2,3)lactose-deficient fed mice were more resistant to DSS-induced colitis than germ-free
mice reconstituted with standard intestinal microbiota. Our study shows that the contact to milk
sialyl(2,3)lactose during infancy affects the bacterial colonization of the intestine, thereby influencing the
susceptibility to DSS-induced colitis in adult mice.
Funded by: Zurich Center for Integrative Human Physiology, Nestlé
13
Nursing our Microbiota: Interactions between Bifidobacteria and Milk Oligosaccharides
DAVID A. MILLS
Department of Viticulture and Enology, University of California Davis, Davis, California, USA
Bifidobacteria are commonly used as probiotics in dairy foods. Select bifidobacterial species are also early
colonizers of the breast-fed infant colon, however the mechanism for this enrichment is unclear. We have
previously shown that Bifidobacterium longum ssp. infantis is a prototypical bifidobacterial species that can
readily utilize human milk oligosaccharides as a sole carbon source. Mass spectrometry-based glycoprofiling has
revealed that numerous B. infantis strains preferentially consume small mass oligosaccharides, abundant in
human milks. Genome sequencing revealed that B. infantis possesses a bias towards genes required to utilize
mammalian-derived carbohydrates. Many of these genomic features encode enzymes that are active on milk
oligosaccharides including a novel 40-kb region dedicated to oligosaccharide utilization. Biochemical and
molecular characterization of the encoded glycosidases and transport proteins have further resolved the
mechanism by which B. infantis selectively imports and catabolizes milk oligosaccharides. Expression studies
indicate that many of these key functions are only induced during growth on milk oligosaccharides and not
expressed during growth on other prebiotics. In addition, key cell surface oligosaccharide binding proteins in B.
infantis bind both milk oligosaccharides and epithelial cell surface glycans. Moreover, growth on milk
oligosaccharides results in significant increases in binding of B. infantis to intestinal cells in vitro. Additional
sequencing of numerous B. infantis isolates has confirmed that these genomic features are common among the
infantis subspecies and likely constitute a competitive colonization strategy employed by these unique
bifidobacteria. By detailed characterization of the molecular mechanisms responsible, these studies provide a
conceptual framework for bifidobacterial persistence and host-interaction in the infant gastrointestinal tract
mediated, in part, through consumption of human milk oligosaccharides.
14
Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides
MOTOMITSU KITAOKA
National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 3058642, Japan
Galactosyl-β1,3-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which was first found in a cell free
extract of Bifidobacterium bifidum, catalyzes the reversible phosphorolysis of galacto-N-biose (Galβ1,3GalNAc,
GNB) and lacto-N-biose I (Galβ1,3GlcNAc, LNB). During the cloning of lnpA gene encoding GLNBP from
Bifidobacterium longum subsp. longum, we found a gene cluster encoding the intracellular pathway specific to
GNB and LNB, the GNB/LNB pathway, involving N-acetylhexosamine 1-kinase, UDP-glucose-hexose/HexNAc 1phosphate uridilyl transferase, and UDP-glucose/GlcNAc 4-epimerase as well as GLNBP. Both the galactose and
N-acetylhexosamine parts of GNB and LNB are converted into the compounds to be entered the glycolytic
pathway. Genes encoding the components of GNB/LNB specific transporter are placed upstream of the genes
encoding these enzymes.
Since oligosaccharides containing LNB in their structure (type I) are predominant in HMOs, the possession of
extracellular enzymes that liberate LNB from HMOs by bifidobacteria explains how HMOs promote the
bifidobacterial growth. Such extracellular enzymatic system of B. bifidum has been identified including α1,2fucosidase, α1,3/4-fucosidase, sialidase, and lacto-N-biosidase.
Bifidobacterial enzymes related to their metabolism of HMOs are useful tool for preparing compounds related to
HMOs. For instance LNB and GNB were produced from sucrose and GlcNAc/GalNAc in one-pot using four
bifidobacterial enzyme including GLNBP. From 10 L of the reaction mixture, 1.4 kg of LNB was isolated as the
crystals. The procedure does not contain any chromatographic techniques and is ready to be scaling-up.
LNB promote growth of particular strains of bifidobacteria that possess GLNBP gene, but not effective to other
bifidobacteria and most lactic acid bacteria. Most bifidobacterial strains that are usually isolated from feces of
infants possess the gene, suggesting the function of the LNB structure in HMOs for the growth of bifidobacteria in
intestines of breast-fed infants. On the other hand, Bifidobacterium adolescentis, the major habitant in adult
intestine, does not have GLNBP and does not utilize LNB.
Funded by: The Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry
15
Human milk oligosaccharides alter human faecal microbiota during in vitro fermentation
ZHUO-TENG YU AND DAVID S. NEWBURG
Higgins hall - Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA
Aims:
The effect of the human milk oligosaccharide fraction (HMOS) on infant fecal microbiota cultured in vitro was
investigated to determine the prebiotic activity of HMOS.
Methods:
Slurries of infant feces were cultured in the presence or absence of HMOS. Differences in the resulting microbiota
culture were measured.
Results:
HMOS supplemented cultures developed significantly lower pH than their unsupplemented negative controls,
and even lower than fructooligosaccharide (FOS) supplemented positive controls. After fermentation, HMOS
supplemented cultures contained higher lactic acid concentrations than the negative control cultures. HMOS
supplemented microbiota contained significantly more Bifidobacteria and Lactobacillus sp. HMOS
supplementation significantly decreased the number of E. coli, Clostridium perfringens, and Clostridium difficile. In
vitro adhesion assays in the presence and absence of HMOS in the medium demonstrate that HMOS significantly
reduces binding of C. perfringens to Caco-2 cells relative to untreated cells, and relative to binding by symbionts
(e.g., bifidobacteria) in the presence of HMOS.
Conclusions:
HMOS supplementation of infant fecal bacteria in culture stimulate growth of Bifidobacteria and Lactobacilli, and
inhibit the growth of clostridial pathogens. HMOS also inhibit binding by C. perfringens to intestinal epithelial
cells. These changes in microbiota community composition are highly reminiscent of differences in microbiota of
breastfed infants relative to microbiota of prematurely weaned infants.
Significance:
These data suggest that the characteristic breastfed microbiome may be attributed largely to the HMOS fraction
of human milk, and that the HMOS fraction of human milk is highly prebotic.
Keywords：human microbiota; HMOS; prebiotics; in vitro fermentation; cell adhesion
16
Characterization of Glycosyl Hydrolases in Bifidobacterium longum subsp. infantis active on
Human Milk Oligosaccharides
DANIEL GARRIDO1,5,6,7, DAVID A. SELA2,5,6,7, SHUAI WU3,5,6, ROGELIO JIMENEZ-ESPINOZA4,7, HYUN-JU EOM2,5,6,7, J.
BRUCE GERMAN1,5,6,7, DAVID E. BLOCK2,4,7, CARLITO B. LEBRILLA3,5,6,7 AND DAVID A. MILLS2,5,6,7
Departments of 1Food Science and Technology, 2Viticulture and Enology, 3Chemistry and 4Chemical Engineering,
University of California Davis, CA USA; 5Foods for Health Institute, University of California Davis, 6Functional
Glycobiology Program, University of California Davis, 7Robert Mondavi Institute for Wine and Food Sciences,
University of California Davis, USA
Human milk provides the infant with several protective mechanisms that encourage its proper development and
growth. Human milk contains a high concentration of complex oligosaccharides, which putatively serve as
prebiotics, favoring the growth of specific members of the infant gut microbiota. As expected, bacteria residing in
this environment have evolved molecular mechanisms for the utilization of these complex substrates. This
includes transport mechanisms and glycosyl hydrolases active on complex oligosaccharides. Bifidobacterium
longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota previously
characterized by its ability to consume several oligosaccharides found in human milk. The genome of this
microorganism revealed various enzymatic systems potentially active on complex host-derived oligosaccharides,
including -fucosidases, -hexosaminidases and -galactosidases. Five genes encode for -fucosidases in B.
infantis. Among these, Blon_0248, Blon_0426 and Blon_2336 were specific for 1-3-fucose linkages, while
Blon_2335 was active on substrates containing 1-2 fucose. The latter two genes were induced by growth on
HMO or lacto-N-tetraose, the most abundant isomer in HMO. Among five genes encoding -galactosidases,
Blon_2016 was induced by LNT and was specific for Galb1-3GlcNAc linkages, abundant in type 1 HMO. On the
other hand Blon_2334 was active on Galb1-4GlcNAc, motif characteristic of type 2 HMO. Both enzymes showed
high Kcat values using ONPG. On the other hand, three -glucosaminidases showed activity on HMO with a
general -hexosaminidase activity, but with different kinetic efficiencies. One enzyme, Blon_2355, was induced
by HMO isomers such as LNT. Finally, these results provide a more complete picture of how B. infantis is able to
consume host milk oligosaccharides, as well as it identifies a number of genes likely associated with
consumption of host glycans.
17
Increased adherence of Bifidobacterium longum subsp. infantis to HT-29 cells following exposure
to a predominant human milk oligosaccharide
DEVON KAVANAUGH1,2,3, LOKESH JOSHI2,3, MARIAN KANE2,3 AND RITA M. HICKEY1,3
1Teagasc
2National
Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
3Alimentary
Glycoscience Research Cluster, Ireland
The intestinal microbiota is vital to human health and nutrition as evident by its contributions to nutrient supply
through degradation of non-digestible food components as well as through the provision of colonisation
resistance to enteropathogens and modulation of mucosal immunity. Bifidobacteria are a member of the
dominant microbiota and can represent up to 80% of cultivable bacteria in infants and 25% in adults.
Bifidobacteria have demonstrated a capacity to digest human milk oligosaccharides (HMOs) via specific
adaptations, possess the ability to influence pro-inflammatory immune responses, and have been assessed for
their efficacy in the treatment and prevention of many human and animal gastrointestinal disorders.
Furthermore, a recent study employing human milk, in which the fat content was removed; demonstrated that
growth of a specific strain of Bifidobacterium in the treated human milk resulted in an increased genetic
expression of putative type II binding fimbriae which are implicated in bacterial colonisation. As the majority of
oligosaccharides in breast milk are able to traverse the GI tract and reach the colon undigested, perhaps HMOs
may contribute not only to selective growth of commensal bacteria, but to their specific adhesive and colonising
ability, as well. In the current study, selected human-milk derived oligosaccharides and commercially available
prebiotics were assayed for their ability to promote the adhesion of Bifidobacterium to the human intestinal cells.
Using these human cell monolayers, we found that pre-incubation of Bifidobacterium with some HMOs
significantly enhanced bacterial adhesion compared to other treatments. Putative mechanisms of how HMOs
influence host-commensal interactions were investigated and will be presented in the poster.
18
Strategies for Screening and Structural Characterization of HMOs
DENNIS BLANK1, SABINE GEBHARDT2, KAI MAASS1, CLEMENS KUNZ2 AND RUDOLF GEYER1
1Institute
of Biochemistry, Faculty of Medicine, Justus Liebig University of Giessen, Giessen, Germany
2Institute
of Nutritional Science, Justus Liebig University of Giessen, Giessen, Germany
Human milk oligosaccharides (HMOs) represent a highly heterogeneous class of carbohydrates which are widely
accepted to be beneficial for human milk fed infants because of their assumed anti-inflammatory, anti-infective
and immune stimulating properties. The structural heterogeneity of HMOs strongly depends on the expression of
specific glycosyltransferases in correlation to the mother’s Lewis blood group and secretor status. Hence,
detailed information on the structural features of these carbohydrates is a prerequisite for future studies on
potential biological functions of these glycans.
In order to facilitate structural and/or compositional assignments we have established an experimental protocol
for mass spectrometric profiling of HMOs starting from fifty microliters of human milk only which allows a rapid
screening of milk samples from different individual donors. Moreover, diagnostically relevant fragment ions of
selected key compositional species could be identified by tandem mass spectrometry enabling a correlation
between the respective HMO pattern and the Lewis blood group of the mother. Based on this information
conclusions on the expression of specific carbohydrate epitopes for each individual milk sample can be easily
drawn without the need of a blood sample.
Characterization of novel HMO structures, however, still requires a different strategy and additional analytical
tools. To this end glycan fractions were fluorescently tagged with 2-aminobenzamide, separated by 2dimensional HPLC and analyzed in native as well as permethylated state by different mass spectrometric
techniques. In addition, linkage positions of individual monosaccharides were assigned by digestion with specific
exoglycosidases or chemical defucosylation in conjunction with GC/MS linkage analysis. Application of this
approach allowed the complete structural characterization of two novel HMO isomers, i.e., a difucosylated
octaose and a trifucosylated decaose.
19
High throughput analysis and quantitation of free oligosaccharides
CARLITO B. LEBRILLA
University of California, Davis, CA, USA
Free oligosaccharides in human milk are involved in several functions necessary for the infant’s health and
development. A hallmark of these compounds is the large structural diversity, which is compounded by the
complicated structures typical of oligosaccharides. For this reason research in this area has been hampered by
the lack of rapid methods for structural analysis. Research in our laboratory is focused at developing methods
for the rapid analysis and quantitation of milk oligosaccharides. While estimates of the number of
oligosaccharides range into the tens of thousands, nanoflow liquid chromatography analysis of milk
oligosaccharides suggests there are only a few hundred structures. A library of structure is being developed by
systematically determining the structures of a number of oligosaccharides. Furthermore, nanoLC with tandem
MS provides a rapid method for structural identification of known structures. In this way, hundreds of
structures can be monitored with complete structures of nearly 100 known. By having a library of known
structures, biological questions are examined including the role of oligosaccharides as prebiotics and as
pathogen blocks.
20
Quantifying human milk oligosaccharide-protein interactions in vitro using electrospray
ionization mass spectrometry
AMR EL-HAWIET, GLEN SHOEMAKER, ELENA N. KITOVA, RAMBOD DANESHFAR AND JOHN S. KLASSEN
Department of Chemistry, University of Alberta, Edmonton, AB, Canada
The direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a powerful tool for
quantifying the association constants for protein-carbohydrate interactions in vitro. The assay is based on the
direct detection and quantification of free and ligand-bound protein ions by ESI-MS for solutions of known initial
concentrations of protein and ligand. The technique boasts a number of strengths, including its simplicity (no
labeling or immobilization required), speed (measurements can usually be completed within 1-2 min), and the
unique ability to provide direct insight into stoichiometry and to study multiple binding equilibria
simultaneously. Additionally, when performed using nanoflow ESI, which operates at solution flow rates in the
nL/min range, the ESI-MS assay affords high sensitivity, normally consuming picomoles or less of analyte per
analysis. A brief overview of the ESI-MS assay will be presented, along with recent methodological advances that
overcome the major sources of error in the binding measurements. Several examples illustrating the application
of the assay for quantifying binding between bacterial proteins (e.g., toxins and adhesins) and human milk
oligosaccharides will be given. A new high-throughput ESI-MS approach for screening carbohydrate libraries
against target proteins will also be described. The “catch and release” ESI-MS assay involves incubating the
target protein with the library of carbohydrates, detecting the protein-carbohydrate complexes by ESI-MS,
activating the complexes to release the carbohydrate ligand, followed by fragmentation of the ligand. The
identification of specific ligands is based on the measured molecular weight and the fragmentation spectrum.
Collision cross section measurements also aid in ligand identification. Several examples highlighting the
application of the “catch and release” ESI-MS assay for the discovery of carbohydrate ligands for a variety of
bacterial proteins, including Clostridium difficile toxins A and B, will be presented.
21
Lewis Blood Group Detection from Human Milk Oligosaccharide Mass Finger Prints
DENNIS BLANK1, KAI MAASS1, VIKTORIA DOTZ2, SABINE GEBHARDT2, CLEMENS KUNZ2 AND RUDOLF GEYER1
1Institute
of Biochemistry, Faculty of Medicine, Justus Liebig University of Giessen, Friedrichstrasse 24, 35392
Giessen, Germany
2Institute
of Nutritional Science, Justus Liebig University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany
The structural diversity of human milk oligosaccharides (HMOs) strongly corresponds with the Lewis (Le) blood
group status of the individual donor. The three different Lewis blood groups, i.e., Le(a−b+), Le(a+b−) and
Le(a−b−) exhibit different expression levels of fucosyltransferases which result in a great structural variety, in
particular, in the case of neutral fucosylated HMO species. These differences in the oligosaccharide patterns are
the basis for our mass spectrometric approach which allows us to assign a milk sample to one of the three
groups. Starting from fifty microliters of human milk the established method provides a time and material saving
way for a Lewis blood group classification of milk samples. The relative abundance of diagnostically relevant
compositional species, such as, Hex2Fuc2, Hex3HexNAc1Fuc2 and Hex4HexNAc2Fuc3 in MS profile spectra is used
as a first step for the identification. In a second step MS/MS analyses of characteristic precursor ions are
performed. For each Lewis blood group, specific mass profiles and fragment ion patterns could be identified
allowing a rapid classification without the need of a blood sample. Furthermore, the outlined protocol can be
used for rapid screening in clinical studies to gain detailed information about neutral as well as acidic
oligosaccharide patterns of specific samples, allowing also a relative quantification of individual compositional
glycan species. Moreover, the described analytical approach enables an easy quality control of milk samples
acquired from milk banks.
22
The development of an annotated structure library for human milk oligosaccharides
SHUAI WU1, NANNAN TAO1, RUDI GRIMM2, J. BRUCE GERMAN3 AND CARLITO B. LEBRILLA1
1Department
of Chemistry, University of California, Davis, CA, USA . 2Agilent Technologies, Palo Alto, CA, USA
3Department
of Food Science and Technology, University of California, Davis, CA, USA
Key Words:
Human Milk Oligosaccharides; Structure Library; Mass Spectrometry; Exoglycosidase Digestion.
Background and Significance:
Human milk oligosaccharides (HMOs) are known as prebiotics stimulating the growth of beneficial intestinal bacteria, and as
receptor analogs inhibiting the binding of pathogens with cell surface glycans. In addition, the development of a balanced
intestinal microflora system may play an important role in modulating the postnatal immune system. Furthermore,
HMOs involved in intestinal absorption and renal excretion may also enhance mineral absorption and promote
postnatal brain development. Since the absorption, metabolism, and function of oligosaccharides have a strong correlation
with their structures, a better understanding of HMO structures will provide important insights into their biological
functions. Almost all HMOs have a lactose core at the reducing end, and they show heterogeneity in terms of composition,
linkages, and branching resulting in a large variety of structures. Elucidating the structures of HMOs has remained a
formidable challenge.
Mass spectrometry provides the most sensitive and rapid method for characterizing HMOs. The combination of nanoflow liquid chromatography (nano-LC) and mass spectrometry (MS) provides orthogonal dimensions involving retention
times, accurate masses, and tandem MS. A HMO structure library has been constructed and annotated using nanoLC/MS/MS system. Together with Agilent database software, the HMO library serves as a novel tool for milk research as
it enables users to identify oligosaccharides very easily and quickly by retention time and mass spectral data. Moreover, it
provides an important reference to study the secretor and Lewis status of the mother and also the absorption and
metabolism of HMOs in their offspring.
Methods and Results:
The HMOs were extracted from pooled milk sample, then reduced with NaBH4 solution, and followed by desalting with solid
phase extraction. Sample profiling was performed on the HPLC-Chip/TOF MS instrument, which separates HMOs on a nanoLC column integrated in a micro-chip. The results generated retention times, accurate masses, monosaccharide
compositions, and relative abundances for 224 HMOs. Thirty-two standard milk oligosaccharides were obtained from
commercial sources and introduced into the Chip/TOF under the identical condition. The corresponding structures in the
milk pool were determined by matching the retention time and accurate mass against the standards. For de novo structural
analysis, the sample was separated into fractions using standard HPLC. Each fraction was analyzed by MALDI FT-ICR and
Chip/TOF to identify the major oligosaccharides in the fraction. IRMPD in the FT-ICR were introduced to study the
branching and connectivity of the structures. Chip/QTOF provides nano-LC separation with online MS/MS. The
collision energy applied was scaled to the size of the oligosaccharides with higher collision energy for larger HMOs. The
fragmentation of isomers under identical collision energy generated different tandem mass spectra. The spectra were distinct
and could be used to identify the oligosaccharides in a different sample. The exact linkages were determined by employing
sequential exoglycosidase digestion. So far, 75 known HMO structures yielding a total of over 200 entries are included in the
library and input into Agilent database software. Accurate mass, retention time, and MS/MS spectra are used to identify all
the oligosaccharide entries.
Funded by: NIH R0 HD061923, NIH R01 HD059127, NIH R01 GM049077
23
Blood Group Dependence of Cholera Infections
ÅSA HOLMNER1,2, ALASDAIR MACKENZIE1 AND UTE KRENGEL1
1Department
2Current
of Chemistry, University of Oslo, Norway
address: Department of Biomedical Engineering & Informatics, Västerbotten County Council, SE-901 85
Umeå, Sweden
The cholera toxin from Vibrio cholerae and the heat-labile enterotoxin from Escherichia coli are major virulence
factors causing cholera and the milder traveler’s diarrhea, respectively. The two toxins share extensive structural
and functional similarities, but they also display distinct characteristics, which find an expression in their
biological function. My group is investigating these two toxins by protein crystallography and complementary
methods. We are interested in elucidating the molecular determinants of these toxins, which determine their
specific characteristics. A question that we have recently become especially interested in is the origin of the
blood group dependence of many infectious diseases, and in particular the molecular basis of cholera blood
group dependence (Holmer et al., 2010). Since human blood group antigens are very similar in structure to
human milk oligosaccharides, breast feeding might have a more direct beneficiary effect by binding inhibition, in
addition to stimulating immunological protection.
In response to increases in temperature, sea level and precipitation variability in already exposed regions of the
world, water-borne diseases are likely to become an increasing threat. The identification of individuals at
particular risk for severe disease will hence become increasingly important for crisis minimization. Furthermore,
understanding of the molecular basis of blood group dependence may pave the way to the development of novel
intervention tools tailor-made for those groups of the population that are at highest risk.
Å. Holmner, A. Mackenzie & U. Krengel (2010). Molecular basis of cholera blood-group dependence and implications for a world
characterized by climate change. FEBS. Lett. 584, 2548-2555.
Funded by: University of Oslo
24
Sialic acid utilization
NORBERT SPRENGER
Nestlé Research Center, Lausanne, Switzerland
Postnatal mammalian development encounters milk as a key environmental variable and yet the sole nutrient
source. Milk is generally considered to have co-evolved with the developmental needs of the suckling newborn.
One evolutionary conserved constituent of milk is sialic acid generally displayed on glyco-conjugates and free
glycans. During postnatal development high sialic acid need was proposed to be unmet by the endogenous sialic
acid synthetic capacity. Hence, milk sialic acid was proposed to serve as conditional nutrient for the newborn.
Using a neonatal rat model, we revisited the relationship between the sialic acid content of milk and sialic acid
metabolism in gut, liver and brain, the major sites of sialic acid synthesis and display. At the other end of
ontogeny, reduced sialylation in brain, saliva and immune system is observed in the elderly. Thus, in analogy to
the neonates the hypothesis that endogenous synthetic capacity cannot keep up with the need in this age group.
Using an aged rat model, we explored sialic acid display and synthesis in gut, brain and liver, and further
investigated the enteric nervous system and stimulated salivation function to assess neuronal function. The data
propose a functional dietary role of sialic acid as a building block for sialylation and beyond.
25
Nutritional Significance of Sialic Acid in Human Milk: an Essential Nutrient for Brain
Development and Cognition
BING WANG1,2,3
1Human
Nutrition Unit, University of Sydney, NSW 2006 Australia
2School
of Medicine, Xiamen University, Xiamen 361005, P. R. China
3Nestlé
Research Centre Beijing, P. R. China
Sialic acid, a family of 9-carbon acidic sugar molecules, are key monosaccharide units in brain gangliosides and
glycoproteins, including the polysialic acid (polySia) glycotope on neural cell adhesion molecules (NCAM).
Human milk is one of nature’s richest sources of sialic acid (~1 g/L) and cow’s milk based infant formulas
contain very little amounts (0~0.25g/L) (1). Gangliosides and polysialylated NCAM in the brain have an
important role in cell-to-cell interactions, neuronal outgrowth, modifying synaptic connectivity, and memory
formation. The alpha 2,8 sialyltransferase IV (ST8SiaIV) is one of two key enzymes for synthesizing polySia on
NCAM. In rodents, the level of NCAM polysialylation increases with learning behavior. The liver can synthesise
sialic acid from glucose, but the activity of the limiting enzyme, UDP-N-acetylglucosamine-2-epimerase (GNE) is
low during the neonatal period. An exogenous source of sialic acid may be critical under conditions of extremely
rapid brain growth, particularly during the first months after birth. We have now demonstrated that dietary
sialic acid supplementation increased the levels of sialic acid in neural tissues, leading to enhanced learning and
memory in piglets, and to up-regulate expression of two learning related genes, Gne and ST8SiaIV (1,2). Global
gene transcription profiling also supports dietary sialic acid supplementation has effects on brain development
and cognition in piglets. We have also found that sialic acid concentration in the frontal cortex of breastfed
infants is higher than the levels of formula-fed infants (1). Taken together, milk sialic acid may be a limiting
nutrient in the neonatal period, facilitating optimal cognitive development in young animals. An exogenous
source of sialic acid enhances brain development, providing a mechanism to explain the link between breastfeeding and higher intelligence.
References:
1) Wang B. Annu. Rev. Nutr 2009;29:177.
2) Wang B et al. Am J Clin Nutr. 2007;85:561-9.
26
Human Milk Oligosaccharides in Amebiasis and Necrotizing Enterocolitis
EVELYN JANTSCHER-KRENN1,2, MONICA ZHEREBTSOV2, TINEKE LAUWAET3, CAROLINE NISSAN1,2, KERSTIN GOTH4,
YIGIT GUNAR4, ANATOLY GRISHIN4, HENRI FORD4, SHARON REED3, FRANCES GILLIN3 AND LARS BODE1,2
1Division
of Neonatology, 2Division of Gastroenterology and Nutrition, Department of Pediatrics, 3Department of
Pathology, University of California, San Diego, 4Saban Research Institute, University of Southern California, USA
Human Milk Oligosaccharides (HMO) are highly abundant in human milk but not in infant formula, which
triggers the questions whether and how HMO benefit the breast-fed infant. Breast-fed infants are at lower risk to
develop such devastating disorders as amebiasis or necrotizing enterocolitis (NEC), and we hypothesized that
HMO contribute to the beneficial effects of breast-feeding in the context of these disorders.
Amebiasis, caused by the protozoan Entamoeba (E.) histolytica, is the third leading cause of death by parasitic
diseases, surpassed only by malaria and schistosomiasis. Worldwide, approximately 50 million people are
infected with E. histolytica, resulting in nearly 100,000 deaths annually. E. histolytica resides in the colon where
it uses a specific Gal/GalNAc lectin to attach to and destroy the host’s epithelial cells. Here, we show that specific
HMO but also Galactooligosaccharides (GOS) prevent E. histolytica attachment and cytotoxicity. These results
suggest that HMO contribute to the lower incidence of amebiasis in breast-fed infants compared to formula-fed
infants. The results also suggest the use of safe, inexpensive and readily available GOS as a novel agent to treat or
even prevent amebiasis.
Necrotizing Enterocolitis (NEC) is one of the most common and often fatal intestinal disorders in preterm
infants. Almost 10% of very-low-birth-weight infants (<1,500g birth weight) develop NEC. More than 25% of
them die from the disorder, and the survivors are often faced with long-term neurological impairment. Breastfed infants are at a 6- to 10-fold lower risk to develop NEC, and we hypothesize that HMO contribute to these
protective effects of breast-feeding. We tested this hypothesis in a rat NEC model and found that HMO
significantly reduced NEC incidence and severity and improved survival. GOS, however, had no effect on NEC.
Moreover, we identified a single HMO that contributed to the protective effects. How HMO protect from NEC
remains unknown.
Funded by: NIH R00 DK078668
27
Interactions of milk glycoconjugates with siglecs
HENDRIK KOLIWER-BRANDL1, NADJA SIEGERT2, ALEXANDER TOLKACH2, ULRICH KULOZIK2 AND SØRGE KELM1
1Centre
for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University Bremen, 28334
Bremen, Germany. 2Institute for Food Process Engineering and Dairy Science, Technische Universität München,
Weihenstephaner Berg 1, 85354 Freising-Weihenstephan, Germany
Siglecs are a family of sialic aid-binding immunoglobulin-like lectins occurring mainly on cells of the immune
system. Their main functions are probably in the regulation of processes leading to the activation of these cells [1].
Consistent with this hypothesis is the observation that most siglecs have tyrosine-based inhibitory motifs
(ITIMs) within their cytoplasmic domains. Probably their sialic acid-binding activities play important roles in
these processes. Of particular interest in this context is the interplay of cis-interactions with glycoconjugates on
their own cell surfaces with trans-interactions with glycoconjugates on opposing cells and in the extracellular
space. Therefore, sialylated compounds like oligosaccharides, glycoproteins and glycolipids like those occurring
in human and animal milk have the potential to modulate siglec-regulated activation processes in the immune
system.
We have used ELISA-like hapten-inhibition assays[2] to investigate the interactions of milk glycoconjugates
with Siglec-2, preferentially binding to 2,6-linked sialic acids, and with Siglec-4, which binds preferentially to 2,3linked sialic acids. As possible sources for bioactive sialylated structures we looked at complex and crude
mixtures from milk fractionation processes. The complementary linkage specificities of these lectins allow the
determination of both 2,3- and 2,6-linked sialic acids even in the presence of high lactose concentrations, as they
are common in milk products[3]. Using these assays, the presence of bioactive sialoglycoconjugates has been
determined in fractions from bovine milk.
Whereas whole milk from local dairy showed no inhibition, samples from milk fractionation processes like
buttermilk, skim milk, lactose-reduced serum, whey proteins as well as caseins and GMP (glycomacropeptides)
were significantly inhibitory, indicating a higher Sia content in these samples. A loss of inhibitory, active
sialoglycoconjugates during fractionation processes was observed for buttermilk samples treated at different
temperatures. Particularly, low pH-values after heat treatment appear to be possible reasons for loss of activity.
In summary, the interaction with Siglec-4 was less sensitive to temperature- and pH-treatment of the samples,
whereas Siglec-2 has lost its inhibition potency completely. The loss of Siglec-2 binding activity could be caused
either by a selective hydrolysis of the α2,6-linkage of sialic acids or by conformational changes in the
glycoconjugate carrier molecules leading to sterically less favored presentations of sialylated glycans serving as
recognition determinants.
[1]Crocker
[2]Bock,
PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol. 2007 Apr;7(4):255-66.
N., & Kelm, S. (2006). Binding and inhibition assays for Siglecs. Methods in Molecular Biology, 347, 359–375.
[3]Koliwer-Brandl,
H., Siegert, N., Umnus, K., Kelm, A., Tolkach, A., Kulozik, U., Kuballa, J., Cartellieri, S., & Kelm, S. (2011). Lectin
inhibition assays for the analysis of bioactive milk sialoglycoconjugates. International Dairy Journal, in press.
Funding by: German Federal Ministry for Education and Research (BMBF, project BioChangePLUS 031632A), the Tönjes-Vagt
Foundation (project XXI)
28
Protein-sugar interactions between secreted fluids and pathogens as a protective mechanism
JASMINE GRINYER1,2, WAI YUEN CHEAH1,2, DANIEL KOLARICH1,3 AND NICOLLE PACKER1,2
1Department
of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW, Australia 2109
2Biomolecular
3Max
Frontiers Research Centre, Macquarie University, Sydney NSW, Australia 2109
Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Glycoproteomics Group,
Arnimallee 22, 14195 Berlin, Germany
The oral environment, comprising the mouth, teeth, tongue and buccal cells, is home to many different bacterial
species. Streptococcus gordonii is an early coloniser of tooth enamel, whereas S. mutans colonises later to cause
dental caries. Bacterial lectins play a role in the initial attachment and biofilm formation by binding to
glycoproteins on the saliva-coated enamel of teeth. The human innate immune system has several mechanisms
for preventing bacterial colonisation on teeth. One such mechanism is thought to be provided by the
glycoproteins in saliva and human milk by protecting the oral cavity from streptococcal species by binding to and
removing these bacteria when swallowed. The removal of bacteria from the oral environment is thus in constant
competition with bacterial binding and infection of the oral cavity.
We have developed two techniques to monitor bacterial binding, one using fluorescent microscopy to visualise
bacterial attachment to saliva-coated hydroxyapatite, and a fluorescent assay on PVDF membrane in a 96-well
plate format to quantitate streptococcal binding to proteins from saliva and human milk. We found that S.
gordonii binds better to saliva and milk proteins/glycoproteins than S. mutans, and S. gordonii binding is
reduced after treatment with neuraminidase to remove the sialic acid residues from the glycoproteins. Structural
determination of saliva and milk protein glycosylation and confirmation of the removal of sialic acids by
neuraminidase was conducted using LC-ESI-MS. Free sialic acid was also shown to modify streptococcal
adherence to saliva and milk. We have shown that sialic acid residues are involved in S. gordonii binding (but not
S.mutans) to saliva and milk glycoproteins, indicating that these secretions can act as a decoy to protect the oral
environment against infection.
Funded by: Macquarie University
29
NOTES
30
SHORT BIOGRAPHIES OF INVITED SPEAKERS
Short Biographies of Invited Speakers
CLEMENS KUNZ – Institute of Nutritional Science, University of Giessen, Germany
Clemens Kunz received his graduate degrees in nutrition from the University of Bonn where he did his
PhD on vitamin D and metabolites in milk at the Department of Pediatrics in 1984. He then joined the
group of Prof. Heinz Egge at the Institute of Physiological Chemistry getting his first scientific contact
with HMOs and has been fascinated by these unique components since then. Subsequently, he held a
postdoctoral fellowship at the University of California, Davis, USA from 1986 to 1989 in Prof. Bo
Lönnerdal’s group focusing on milk proteins and glycoproteins. He then became leader of the working
group Clinical Chemistry at the Research Institute for Child Nutrition in Dortmund and a Professor of Physiological Chemistry at
the University of Bonn, continuing his research in the field of HMOs with a focus on metabolic aspects. In 1999 he was appointed
Professor of Human Nutrition at the Justus-Liebig-University of Giessen. His current research focuses on structural, functional
and metabolic aspects of HMOs including the use of stable isotopes and on the bioavailability and metabolism of polyphenols
from berries in healthy humans. His work yielded several research awards and has continuously been funded by the German
Research Foundation (DFG), the Federal Ministry of Science and Education (BMBF) and the Hessian Ministry of Science and Arts
(HMWK).
HUDSON FREEZE – Sanford-Burnham Medical Research Institute La Jolla, CA, USA
Hudson Freeze earned his Ph.D. from the University of California at San Diego in 1976. Subsequently
he held fellowships in Biology, Medicine and Neurosciences and later joined the faculty at the same
institution. In 1988, Dr. Freeze was recruited to Sanford-Burnham Medical Research Institute
and served as the Director of the Glycobiology and Carbohydrate Chemistry Program from 20002008. His work focuses on pathology resulting from faulty glycosylation, the process of adding sugar
chains to proteins and lipids. Carbohydrates are required for proper secretion and targeting of thousands of proteins, an often
overlooked fact of biology. He is driven by the search for novel therapeutics to treat patients with mutations leading to
glycosylation defects called Congenital Disorders of Glycosylation or CDGs.
TADASU URASHIMA – Obihiro University, Hokkaido, Japan
Tadasu Urashima was born in Hiroshima at 12/3/1957. Degrees: (1) undergraduate course: Tokyo
University of Agriculture and Technology, 1980. (2) master course: Tohoku University, 1982. (3) Ph.D.
course: Tohoku University, 1986. Past Positions: (1) Assistant Professor at Obihiro University of
Agriculture and Veterinary Medicine, April 1986. (2) Associate Professor at Obihiro University of
Agriculture and Veterinary Medicine, April 1994. (3) Professor at Obihiro University of Agriculture and
Veterinary Medicine, August 2003. Present position: Professor at Obihiro University of Agriculture and Veterinary Medicine,
Graduate School of Animal and Food Hygiene. Studies: (1) 1986-present: Characterization of milk oligosaccharides of domestic
farm animals. (2) 1991: Beta N-acetylglycosaminyltransferase activity in lactating mammary glands of tammar wallaby, Sydney
University, under Dr. Michael Messer. (3) 1995-present: Characterization of milk oligosaccharides of captured wild animals
including primate species, Canoidea species, elephant, Felidae species, Cetacea species etc, with Dr. Olav Oftedal. (4) 2000present: Determination of each human milk oligosaccharide concentration. (5) 2009-present: Determination of each milk
oligosaccharide level in the broth of Bifidobacteria, with Dr. Motomitsu Kitaoka, Dr. Takane Katayama, Dr. Kenji Yamamoto and
Dr. Sadaki Asakuma. (6) 1994-present: Extracellular polysaccharides produced by dairy lactic acid bacteria, with Dr. Bill Bubb
and Dr. Kenji Fukuda. (7) 2006-2010: Bioactive components in bovine colostrum, with Dr. Takashi Terabayashi, Dr. Minoru
Morita and Dr. Kenji Fukuda.
31
SHARON M. DONOVAN – Department of Food Science and Human Nutrition, University of
Illinois, Urbana, IL, USA
Sharon Donovan received her B.S. and Ph.D. in Nutrition from the University of California, Davis. She is
also a Registered Dietitian. After completing a post-doctoral fellowship in Pediatric Endocrinology at
Stanford University School of Medicine, she accepted a faculty position at the University of Illinois,
Urbana in 1991. She was promoted to Professor in 2001 and, in 2003, was named the first recipient of
the Melissa M. Noel Endowed Chair in Nutrition and Health at the University of Illinois. Her research
focuses on pediatric nutrition, with an emphasis on optimization of neonatal intestinal development. She compares the
biological effects of human milk and infant formulas on intestinal function in human infants, neonatal piglets and in various
models of intestinal disease. She has published over 100 peer-reviewed publications, review articles and conference proceedings.
Her research is funded by NIH, USDA and private industry and foundations.
THIERRY HENNET – Institute of Physiology and Center for Integrative Human Physiology,
University of Zurich, Switzerland
Thierry Hennet is a Professor in the Institute of Physiology and Center for Integrative Human
Physiology at the University of Zurich. His research interest is on Defects of glycosylation that lead to
diseases with variable clinical manifestations, encompassing neurological disorders, congenital
muscular dystrophies, connective tissue disorders, immune deficiencies, and coagulopathie.
His group investigates the roles of specific glycoconjugates and glycosyltransferase enzymes in health and disease. To this end,
they combine the study of genetic animal models to biochemical assays performed in cell culture systems. In addition, he studies
the molecular basis of novel types of Congenital Disorders of Glycosylation (CDG), a family of diseases characterized by abnormal
biosynthesis of several classes of glycoconjugates.
DAVID A. MILLS – Department of Viticulture and Enology, University of California Davis, Davis,
California, USA
David Mills is a Professor in the Department of Viticulture and Enology in the Robert Mondavi Institute
for Wine and Food Sciences at the University of California at Davis. For over 20 years Dr. Mills has
studied the molecular biology of lactic acid bacteria involved in food and beverage fermentations or
active as probiotics in intestinal health. In the last decade, Mills led the Lactic Acid Bacteria Genomics
Consortium which resulted in a seminal comparative analysis and release of key genome sequences of
food-grade lactic acid bacteria and bifidobacteria. More recently with Bruce German and Carlito Lebrilla, Dr. Mills formed the UC
Davis Milk Bioactives Program, a multidisciplinary effort to characterize the influence of milk glycans on intestinal health. Dr.
Mills has served as a Waksman Foundation Lecturer and Chair of the Food Microbiology Division of the American Society for
Microbiology. He currently serves as an associate editor for the journal Microbiology. In 2010 Dr. Mills was awarded the Cargill
Flavor Systems Specialties Award from the American Dairy Science Association.
Dr. Mills obtained a Bachelors of Science in Biochemistry from the University of Wisconsin-Madison. In 1991, he obtained a
Masters degree in Biochemistry with Dr. Michael Flickinger at University of Minnesota, followed by a PhD in Microbiology in
1995 working with Dr. Gary Dunny and Dr. Larry McKay. After postdoctoral studies at North Carolina State University with Dr.
Todd Klaenhammer, Dr. Mills took a faculty position at UC Davis.
32
MOTOMITSU KITAOKA – National Food Research Institute, National Agriculture and Food
Research Organization, Tsukuba, Ibaraki 305-8642, Japan
Motomitsu Kitaoka is a research leader of the enzyme laboratory at the National Food Research
Institute of the National Agriculture and Food Research Organization, Japan. He obtained a Ph. D. from
the University of Tokyo in 1993. After completing a post-doctoral fellowship at the Iowa State
University, he moved to the National Food Research Institute in 1998. He has been working on
carbohydrate degrading enzymes for more than 20 years, especially on sugar phosphorylases. He has
been pursuing practical procedures to produce oligosaccharides, including a large-scale preparation of lacto-N-biose I. He is also
interested in rational mutation to convert glycoside hydrolytic enzymes into catalysts for the syntheses of glycosides.
RUDOLF GEYER – Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University
of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany
Rudolf Geyer studied Chemistry at the University of Darmstadt and the University of Freiburg in
Germany. After graduation he worked for his Ph.D. at the Max-Planck-Institute of Immunology in
Freiburg and received his degree in Biochemistry in 1977 at the University of Freiburg. He then
moved to Giessen, Germany, were he worked as a research assistant and later as a university
assistant at the Institute of Biochemistry of the Giessen University Medical School. In 1990 he was appointed as a professor of
biochemistry and leader of the Glycobiology Unit at the same institute. His present research interests are mainly focused on
structures and putative functions of glycoconjugates from parasitic helminths as well as on the carbohydrate structures of
mammalian and invertebrate glycoproteins and human milk oligosaccharides.
CARLITO B. LEBRILLA – University of California, Davis, CA, USA
Carlito Lebrilla is a Professor at the University of California, Davis in the Department of Chemistry and
Biochemistry and Molecular Medicine at the School of Medicine. He is currently the Chair of the
Chemistry Department. He was born in the Philippines. He received his BS degree from the University
of California, Irvine and Ph.D. from the University of California, Berkeley. He was an Alexander von
Humboldt Fellow and a NSF-NATO Fellow at the Technical University in Berlin. He returned to the UC
Irvine as a President’s Fellow and has been at UC Davis since 1989. His research is in Analytical
Chemistry, primarily mass spectrometry with applications to clinical glycomics and biofunctional food. He has over 225 peerreviewed publications. He is also co-editor of Mass Spectrometry Reviews and has been on the editorial board of Mass
Spectrometry Reviews, Journal of American Society for Mass Spectrometry, European Mass Spectrometry, and International
Journal of Mass Spectrometry.
33
NORBERT SPRENGER – Nestlé Research Center, Lausanne, Switzerland
Norbert Sprenger studied biology at the University of Basel, Switzerland, and received
his B.S. in Developmental Biology and his Ph.D. in Molecular Plant Physiology with Profs.
A. Wiemken and T. Boller for his studies on plant storage, transport and stress
carbohydrates. He then took a first post-doctoral position in plant molecular physiology
in the laboratory of Prof. F. Keller at the University of Zurich, Switzerland. For a second
post-doctoral fellowship, financed by grants from the Swiss National Science Foundation, Novartis and the Human Frontier
Science Program Organisation, he joined the laboratory of Prof. C. Somerville at Stanford University at the Carnegie Institute for
Science department of plant biology, where he studied plant cell wall synthesis and physiology using molecular genetics
approaches. He then switched to mammalian physiology accepting a position as research scientist in Glycobiology at the Nestlé
Research Center in Lausanne, Switzerland. His research focuses on functional glycans for pediatric and medical nutrition, with an
emphasis on intestinal development and barrier function.
BING WANG – Human Nutrition Unit, University of Sydney, Australia; School of Medicine,
Xiamen University, P. R. China; Nestlé Research Centre Beijing, P. R. China
Bing Wang received her M.D. degree from Tianjin Medical University, China, and a Ph.D. in Science
(Biochemistry) from the University of Sydney, Australia. She is a currently an Honorary Associate in the
School of Molecular & Biosciences at the University of Sydney, a Min-Jiang Scholar and Adjunct
Professor of Molecular Medicine, School of Medicine, Xiamen University, China and a Senior Research
Scientist at the Nestlé Research Centre-Beijing. She is an internationally recognized expert in
nutritional glycobiology. She pioneered the development of the piglet as a model system to elucidate the molecular mechanisms
underlying the role of milk sialic acid in brain development and cognition. She is also a registered Nutritionist of the Nutrition
Society Australia. Her studies have been carried out at both the gene and biochemical levels on the functional effect of new food
ingredients.
LARS BODE – Division of Neonatology and Division of Gastroenterology and Nutrition,
Department of Pediatrics, University of California, San Diego, USA
Lars Bode received his M.S. and Ph.D. in Nutrition from the Justus-Liebig University, Giessen, Germany.
After determining structural differences in the lipid composition of human and bovine milk gangliosides
as a Master student, Lars joined Dr. Clemens Kunz’s lab as a PhD student and worked with Dr. Silvia
Rudloff to study the effects of human milk oligosaccharides on selectin-mediated cell-cell interactions in
the immune system. After a predoctoral fellowship at the Institute of Child Health, University College
London, Lars joint Dr. Hudson Freeze’s lab as a postdoctoral fellow in the Glycobiology and Carbohydrate Chemistry Program at
the Burnham Institute for Medical Research. In 2009 the University of California, San Diego, School of Medicine recruited Lars as
an Assistant Professor of Pediatrics in the Division of Neonatology and the Division of Gastroenterology and Nutrition, where his
lab develops a new research program to elucidate functions and biosynthesis of human milk oligosaccharides. Lars is an affiliate
of the Glycobiology Research and Training Center and the Digestive Disease Research Development Center at the University of
California, San Diego. Lars is the Chair of the Lactation Research Interest Group of the American Society for Nutrition and on the
Executive Board of the International Society for Research on Human Milk and Lactation. Lars’ research is supported by an NIH
Pathway to Independence Award, and funded by NIH and private industry.
34
ABSTRACTS OF POSTERS
Abstracts of Posters
Role of Sialic acid in Innate Immune Protection provided by Mammalian Milk
WAI YUEN CHEAH1, KATHERINE WONGTRA-KUL KISH1, DANIEL KOLARICH2, JASMINE GRINYER1, NICOLLE PACKER1
1Department
of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Sydney, NSW,
Australia
2Department
of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Berlin, Germany
Sialic acid contained in mammalian milk has been reported to have an immune protective role for the infant.
Human milk contains a large amount of sialic acid compared with bovine milk, with sialic acids found on
glycoproteins, glycolipids and free oligosaccharides. However, the mechanism by which this monosaccharide
inhibits infection remains unknown. We have investigated how milk confers innate immune protection to the
infant gut through binding of gastrointestinal-associated bacteria and compare the difference in binding of
specific bacteria to human and bovine milk glycoconjugates.
Different methods to fractionate human and bovine milk were explored to determine which fraction contains
bacterial binding glycoconjugates. Milk fractions were adhered to PVDF membranes in a 96 well format.
Fluorescently-labelled bacteria were added to the wells and the binding of the bacteria to the fractions was
quantified by fluorescence. To determine whether the glycans were involved in this interaction, in particular
sialylated glycans, the glycoconjugate structures were altered using exoglycosidases and the effect on bacterial
binding was measured. Exoglycosidase activities were confirmed by glycan profiling of the N- and O-glycans
released by PNGase F and β-elimination, respectively, of milk glycoproteins using graphitised carbon LC-ESIMS/MS. The inhibition of binding of the bacteria to the milk glycoconjugates was tested by pre-incubation of the
bacteria with free oligosaccharides.
We found that specific bacteria bind differently to human and bovine milk glycoconjugates. Most bacteria had
higher binding affinity to the milk protein fraction than the lipid fraction. Bacterial binding was inhibited both by
removal of sialic acid with sialidase and by pre-incubation of the bacteria with sialic acid. This confirms that
sialic acid can competitively inhibit bacterial adhesion to both human and bovine milk glycoconjugates. These
data indicate the importance of sialylated milk glycoconjugates in binding to gastrointestinal-associated bacteria
and providing innate immune protection to the infant’s gut.
Funded by: Macquarie University
35
Ascending colonic microbiota composition and SCFA patterns produced from in vitro
fermentation of human milk oligosaccharides and prebiotics differ between formula-fed and
sow-reared piglets
MIN LI1, LAURA L. BAUER2, XIN CHEN1, MEI WANG1, THERESA KUHLENSCHMIDT3, MARK S. KUHLENSCHMIDT3,
GEORGE C. FAHEY JR.2 AND SHARON M. DONOVAN1
1Department
of Food Science and Human Nutrition, 2Department of Animal Sciences, 3Department of Pathobiology,
University of Illinois, Urbana, IL 61801, USA
The objective was to compare the effect of piglet age and sow-rearing versus formula feeding on in vitro
fermentation characteristics and gut microbial modulation properties of human milk oligosaccharides (HMO),
lacto-N-neotetraose (LNnT), a predominant HMO, and three commonly used prebiotics: a 1:2 mixture of
galactooligosaccharide (GOS) and polydextrose (PDX), and short-chain fructooligosaccharides (scFOS). Ascending
colon contents were obtained from four donor groups: 9 and 17 day-old formula-fed (FF9, FF17) and sow-reared
(SR9, SR17) piglets. The changes in pH and gas, short chain fatty acid (SCFA) and lactate production were
determined following 0, 4, 8, and 12 h of incubation. The pH change and total SCFA, acetate and propionate
production were greater in the FF than SR, and in the 9- compared to the 17-day-old piglets, regardless of diet.
However, SR produced higher amounts of gas, butyrate and lactate than FF piglets. For most donors, the pH
change was greatest for scFOS, and least for GOS/PDX. The fermentation of LNnT produced larger amounts of gas,
total SCFA, acetate, and butyrate than did the other substrates, whereas higher amounts of propionate and lactate
were observed from HMO and scFOS fermentation respectively. Gut microbial populations were assessed by 16S
rRNA V3 gene denaturing gradient gel electrophoresis (DGGE) analysis and group-specific real-time PCR. Global
gut microbial structures differed among four piglet groups prior to fermentation. SR had higher concentration of
Bifidobacterium and Lactobacillus, whereas FF had greater amounts of Clostridium cluster IV and XIVa. The
concentrations of these four bacteria groups were higher in 9- than 17-day-old piglets. Changes in microbial
patterns during fermentation were assessed by DGGE. Bands that increased in density on most substrates were
identified by sequencing as related to Bacteroides vulgatus, Collinsella aerofaciens, Anaerovibrio sp., and species
belong to Clostridium cluster IV, XIVa and XVIII. The concentrations of Bifidobacterium, Clostridium cluster IV and
XIVa and B. vulgatus detected by qPCR increased after 8 and 12h fermentation on most substrates. In summary,
piglet diet and age affect gut microbial populations, which leads to different bacterial fermentation patterns. HMO
and LNnT have potential prebiotic effects due to their ability to be fermented to SCFA and modulate microbial
populations.
Funded by: NIH R01 HD061929
36
Oligosaccharide MALDI-MS profiles in milk and urine from mother-child pairs
VIKTORIA DOTZ1, SILVIA RUDLOFF1,2, DENNIS BLANK3, SABINE GEBHARDT1, KAI MAASS3, RUDOLF GEYER3 AND
CLEMENS KUNZ1
1Institute
of Nutritional Science, University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany
²Department of Pediatrics, University of Giessen, Feulgenstrasse 12, 35392 Giessen, Germany
³Institute of Biochemistry, Faculty of Medicine, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany
Human milk contains a large variety of complex lactose-based oligosaccharides (HMO) in concentrations ranging
from 10 to 20 g/L. The presence of different neutral HMO depends on the activity of specific glycosyltransferases
in the mammary gland. Therefore, the presence of 1-2-, 1-3- and/or 1-4-fucosylated core oligosaccharide
structures is determined by the secretor status and the Lewis (Le) phenotype of the mother (1,2). So far, little
information exists about the metabolic fate of HMO in newborns. In previous studies, we found a renal excretion
of about 1-2 % of the ingested LNT and LNFPII in exclusively breast-fed preterm infants (3). Here, we report on
the excretion profile of HMO in term infants.
The aims of the study are (i) to compare the HMO pattern in milk of exclusively breastfeeding women with the
urinary oligosaccharide profile of their infants and (ii) to investigate whether the infants´ urinary HMO profile
resembles the pattern of Lewis-specific HMO in their mothers´ milk.
After application of an oral 13C-galactose bolus to 8 lactating mothers, milk at each nursing and urine of the
infants was collected for 36 hours (4). For analytical purposes, 50 µL of a single milk sample and 500 µL of the
infant urine sample were used for solid phase extraction prior to MALDI-MS of the non-derivatised HMO fraction.
Lewis blood group determination was performed on all milk samples.
Resulting from their characteristic MS profile as well as MS/MS spectra, the following Lewis phenotypes were
found: Milk from 6 women was Le(a−b+), one sample was Le(a+b−) and one presumably Le(a−b−) or Le(a−b+).
In the corresponding urinary samples of the infants, the same characteristic MS profiles were observed with
regard to relative HMO peak intensities. However, HMO in the lower mass range were more predominant in
urine, particularly for the infant whose mother was Le(a+b−). Moreover, MS/MS data revealed that none of the
Le(a−b+)-specific HMO were detected either in the milk of the Le(a+b−) mother or in the urine of her infant.
MS/MS of urine from infants fed with Le(a−b+)-milk confirmed the presence of Le(a−b+)-specific HMO.
Our data indicate that the HMO patterns in urine from breast-fed infants reflect those from their mothers’ milk
suggesting a strong association to the mothers´ Lewis phenotype determined in milk.
Literature:
1) Kunz et al., Annu Rev Nutr 2000; 2) Thurl et al., Br J Nutr 2010; 3) Rudloff et al. Acta Paediatr 1996; 4) Rudloff
et al., Glycobiol 2006
Funded by: German Research Foundation Ru 529/7-3 and Ku 781/8-3
37
Human Milk Oligosaccharides as Anti-adhesion Candidates for Clostridium difficile Toxin
AMR EL-HAWEIT1, ELENA N. KITOVA1, PAVEL KITOV1, LUIZ EUGENIO2, KENNETH K.S. NG2, GEORGE L. MULVEY3,
TANIS C. DINGLE3, ADAM SZPACENKO1, GLEN D. ARMSTRONG3 AND JOHN S. KLASSEN1
1Department
of Chemistry, University of Alberta, Edmonton, AB, Canada.
2Department
of Biological Sciences, University of Calgary, Calgary, AB, Canada.
3Department
of Microbiology & Infectious Diseases, University of Calgary, Calgary, AB, Canada. †Alberta Ingenuity
Centre for Carbohydrate Science
Human milk oligosaccharides (HMOs) play an important role in protecting neonates from pathogenic
microorganism especially the enteric bacteria. This is believed to be due to their ability to act as soluble
receptors that inhibit the pathogen binding to their host cell target ligands. Clostridium difficile is the leading
cause of hospital acquired diarrhea and pseudomembranous colitis in Europe and North America. The key
virulence determinants of C. difficile are the two exotoxins, toxin A (TcdA) and toxin B (TcdB). Those exotoxins
are believed to bind to carbohydrate receptors on the intestinal epithelium. Here, we describe the first
quantitative study of the binding of HMOs to the C. difficile toxins. The direct electrospray ionization mass
spectrometry (ESI-MS) assay has been applied to determine the binding affinities of recombinant subfragments
TcdA and TcdB of C. difficile toxins against a library of twenty one oligosaccharides representing, the most
common neutral and acidic HMOs. The results of the ESI-MS measurements indicate that both toxins fragments
bind specifically to several HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested
bind to both toxins: Fuc(1-2)Gal(1-4)Glc, Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc, Fuc(1-2)Gal(1-3)GlcNAc(13)Gal(1-4)Glc, Gal(1-3)[Fuc(1-4)]GlcNAc(1-3)Gal(1-4)Glc and Gal(1-4)[Fuc(1-3)]GlcNAc(1-3)Gal(14)Glc. Although, the binding of the HMOs is uniformly weak, with apparent affinities ≤3000 M-1, a number of
HMOs bind to the toxins with a much higher affinity than the only known natural receptor,
Gal(1,3)Gal(1,4)Glc, for which binding constants for both A2 and B1 were found to be ~500 M -1. The results
of the molecular docking study suggest that the general mode of carbohydrate recognition may be conserved
between TcdA and TcdB, with a lactose disaccharide occupying the central portion of the carbohydrate binding
site for both toxins. Analysis of the docked structures also helps to explain how the differences in distributions of
negatively and positively charged side chains in the binding pocket of TcdA and TcdB influence ligand binding
specificity. The Verocytotoxicity neutralization assay reveals that HMO fractions extracted from human milk do
not significantly inhibit the cytotoxic effects of TcdA nor TcdB. The absence of protection is attributed to the
weak intrinsic affinities that the toxins exhibit towards the HMOs.
Funded by: 1. Alberta Ingenuity Centre for Carbohydrate Science (AICCS)
2. Natural Sciences and Engineering Research Council (NSERC)
3. University of Alberta
38
Structure determination of bacterial mucus-binding proteins and their functional role in
adhesion to host glycans
SABRINA ETZOLD1, DONALD MACKENZIE1, LOUISE E. TAILFORD1, ROB FIELD2, ANDREW HEMMINGS3 AND
NATHALIE JUGE1
1Institute
2John
of Food Research, Norwich Research Park, Norwich NR4 7UA, UK
Innes Centre, Norwich Research Park, NR4 7UH, UK
3University
of East Anglia, Norwich NR4 7TJ, UK
The mucus layer covers the epithelial cells of the gastrointestinal (GI) tract and protects the underlying mucosa
from the lumen content. The main structural components of mucus are highly glycosylated mucin proteins
carrying mainly O-linked glycan chains characterised by a high level of structural complexity and diversity.
Protein-carbohydrate interactions are believed to play an important role in the adhesion of resident gut bacteria
to the mucus layer. However, the nature of the ligands and the specificity of the interaction remain to be
elucidated. Our research focuses on lactobacilli mucus-binding proteins (MUB) whose presence on the bacterial
cell-surface contributes to bacterial attachment to the protective mucus layer. MUBs are composed of tandemlyarranged mucus-binding amino acid sequence repeats (Mubs). The 353 kDa MUB from the L. reuteri strain ATCC
53608 consists of two types of repeats, Mub1 and Mub2, present in six and eight copies, respectively. We
determined the first crystal structure of a type 2 Mub repeat (184 amino acids) at 1.8-Å, displaying high
structural similarity to the repeat-unit of the Peptostreptococcus magnus Protein L (PpL), an immunoglobuling
(Ig)-binding surface protein, and we showed that Mub repeats were able to interact with a number of
mammalian Igs in vitro. Our current work is to obtain structural information on type 1 Mub repeats and multirepeat modules to gain further insight into the structural organisation of MUB. In addition, we focus on the
characterisation of the Mub repeat interaction with mucins and mucin glycans, whose structure can be similar to
oligosaccharides found in milk, using a range of biophysical methods including in vitro binding assays,
carbohydrate arrays and Isothermal Titration Calorimetry. Elucidating the molecular basis of host-bacteria
interaction is crucial for the understanding of host colonisation and the beneficial role of lactobacilli in the GI
tract.
Funded by: BBSRC, NRP
39
Breast milk microbiota: is there a relationship with HMOs?
ANA SOTO, NIVIA CÁRDENAS, SUSANA DELGADO, JUAN MIGUEL RODRÍGUEZ AND LEONIDES FERNÁNDEZ
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid,
Avda. Puerta de Hierro, Madrid, Spain
Breast milk is a complex biological fluid that provides all the nutritional requirements for the first months of life
but, additionally, educates the infant immune system and confers protection against pathogens. These effects
result from the synergistic action of many bioactive molecules, such as cytokines, cellular components,
oligosaccharides or lipids. While it is well known that breast milk is rich in oligosaccharides, it has only recently
been accepted that human milk also constitutes a source of commensal and probiotic bacteria which seems to
play an important role in gut colonization and modulation of the infant gut.
In recent years, analyses of the bacterial diversity of human milk have revealed that this biological fluid is an
important source of live staphylococci, streptococci, bifidobacteria and lactic acid bacteria to the infant gut. In
contrast to other bacteria, these seem to be uniquely adapted to reside in the human digestive tract and to
interact with us in symbiosis from the time we are born. Traditionally it was considered that bacteria present in
breast milk were acquired by mere skin contamination. However, it has been found that lactobacilli and
enterococcal isolates present in human milk are genotypically different from those isolated in the skin within the
same host. Furthermore, several studies suggest the existence of a mammary microbiota during late pregnancy
and lactation. Live bacteria from the maternal gut could colonize the mammary gland through an endogenous
route (the so-called entero-mammary pathway), involving maternal dendritic cells and macrophages.
In this context, the global objective of this work was to ascertain if there is a relationship between human milk
oligosaccharides and microbiota. For that purpose, carbohydrate utilization was studied in a variety of lactic acid
bacteria and bifidobacteria isolated from human milk of healthy donors, and compared with their enzymatic
activity on chromogenic substrates. Genome sequences of selected bacteria were analyzed to identify possible
genes involved in the metabolism of carbohydrates, especially those related to HMOs.
Funded by: AGL2010-15420 (2010-2013), RM2009-00009-00-00
40
Tailoring carbohydrates to capture toxins and pathogens
SURI S. IYER
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA
Over the past few years, our group has been harnessing their recognition properties to develop high affinity
ligands for toxins and pathogens. Carbohydrates are excellent recognition molecules and modulate essential
communication processes as cell adhesion, proliferation, migration, differentiation and metastasis. Synthetic
carbohydrates can be used for capturing pathogens as they are highly robust under a variety of conditions,
refrigeration is not required and can be stored for extended period (over months), inexpensive, not subject to
lot-to-lot variation, can be manipulated to suit any sensor platform as these are small molecules and not subject
to antigenic drift because toxicity is intimately associated with the cell binding sites. However, synthetic
carbohydrates have “thought” to suffer from selectivity and sensitivity issues for practical applications. We have
recently demonstrated that it is possible to tailor glycoconjugates to achieve high selectivity and sensitivity
towards specific analytes. We have synthesized a library of tailored carbohydrates using a novel modular
strategy for the rapid production of these molecules (Bioorg. Med. Chem. Lett., 2007, 17, 2459-64). We have
demonstrated that synthetic carbohydrates differentiate between closely related strains of Shiga toxin, (Angew.
Chem. Int. Ed. Engl. 2008, 47, 1265-68; Highlighted in Chemical and Engineering News, 2008, 86, 42) influenza (J.
Am. Chem. Soc. 2008, 130, 8169–71) and Escherichia coli. (Chembiochem, 2008, 9, 2433-42). Our other relevant
publications on the molecular nature of carbohydrate-protein interactions include ChemBioChem, 2009, 10,
1486-89; Biochemistry, 2010, 49, 1649-57; Medicinal Research Reviews, 2010, 30, 327-93; Bioconj. Chem., 2010,
21, 1486-93 and Biochemistry, 2010, 49, 5954-67. Recently, we have developed a simple, rapid, and sensitive
glycan-based magnetic relaxation switch assay for the detection of glycan binding proteins. We demonstrated
that magnetic relaxation switch assays can be used to detect toxins in a complex medium such as stool and
environmental samples. (Anal. Chem., 2010, 82, 7430-7435). Our efforts in this area towards the development of
potential diagnostics and therapeutics using natural and modified oligosaccharides will be the focus of this
presentation.
Funded by: NSF CAREER GRANT, NSF CHE 0845005; NIAID U01 AI075498
41
α-L-Fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains
TAKANE KATAYAMA1, HARUKO SAKURAMA1, YUJI HONDA1, MOTOMITSU KITAOKA2 AND KENJI YAMAMOTO1
1Research
Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-
8836, Japan
2National
Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-
8642, Japan
Lewis a (Lea) and Lewis x (Lex) blood group antigens attached at the non-reducing ends of glycoconjugates
control various pivotal biological events; therefore, the efficient synthesis of the antigens is important to
understand their physiological roles. Here, we show that the nucleophile-mutant D703S of 1,3-1,4-α-L-fucosidase
from Bifidobacterium bifidum exclusively synthesizes Lea and Lex trisaccharides when incubated with β-Lfucopyranosyl fluoride (FucF) as a donor and lacto-N-biose I (Galβ1-3GlcNAc) and N-acetyllactosamine (Galβ14GlcNAc) as acceptors, respectively, with yields of 56% against the added FucF. The synthase also recognized
lactose, 2’-fucosyllactose and lacto-N-tetraose as acceptors, and specifically produced 3-fucosyllactose,
difucosyllactose and lacto-N-fucopentaose II, respectively. In contrast, the enzyme did not accept
monosaccharides, cellobiose, Glc1-4GlcNAc, N,N’-diacetylchitobiose or galacto-N-biose as substrates. The
results revealed that the enzyme specifically recognizes the disaccharide structures [Galβ1-3/4GlcNAc(Glc)] at
the non-reducing ends and attaches a Fuc residue via an α-(1→3/4)-linkage to the GlcNAc/Glc residue. The strict
regio- and acceptor specificity of this 1,3-1,4-α-L-fucosynthase is unique and should serve as a potentially
powerful tool to specifically introduce Lea and Lex epitopes in type-1 and type-2 chains of glycans, respectively.
Funded by: Grant-in-Aid for Scientific Research by Young Scientists (B) 22780072 from Ministry of Education, Culture,
Sports, Science and Technology, Japan
42
A new methodology for screening of bacteria-carbohydrate interactions: anti-adhesive milk
oligosaccharides as a case study
JONATHAN A. LANE1, 2, KARINA MARIÑO3, PAULINE M. RUDD3, STEPHEN D. CARRINGTON2 AND RITA M. HICKEY1
1Teagasc
Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. 2Veterinary Science Centre, University
College Dublin, Belfield, Dublin 4, Ireland. 3Dublin Oxford Glycobiology Group, National Institute for Bioprocessing
Research & Training (NIBRT), Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
Carbohydrates have been shown to inhibit the initial recognition events leading to adhesion and colonization of host
tissues by pathogens and therefore have potential to prevent infection. Some of the most efficient anti-adhesion agents
identified to date are present in foodstuffs - as best exemplified by human milk carbohydrates which protect newborns
against infections. Such carbohydrates can display structural homology to host cell receptors or pathogenic lectins,
thus functioning as receptor decoys. However, the large amounts of human milk carbohydrates which are required for
intervention or clinical studies are unavailable; therefore researchers are beginning to focus their attention on sources
of carbohydrates other than those from human milk. For instance, the milk from domestic animals, honey, fermented
products, commensal and food-grade bacteria all contain carbohydrates which are structurally similar to human milk
carbohydrates and have been in some cases shown to prevent pathogen binding to host cells. However, current
screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming
and require large amounts of oligosaccharides. Therefore, there is a need to develop analytical techniques that can
quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using cell line models of infection.
Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential antiadhesive activity against bacteria. This label-free approach involves the brief and iterative exposure of a mixture of
free oligosaccharide to aliquots of a defined bacterial population. These organisms act as an “affinity matrix” to
progressively deplete glycans with the specific capacity to bind to the surface of these microbes, permitting their
identification through comparison with untreated glycan profiles. As a case study, the free oligosaccharides from the
colostrum of Holstein Friesian cows were screened for interactions with E. coli O157:H7 cells. Reductions in
oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography. The use of the
depletion assay showed selective bacterial interaction with lactose, 3’-sialyllactose, disialyllactose, and
sialyllactosamine in a population dependent manner, in line with previous results obtained by different methodologies.
The depletion assay methodology was validated by inhibition studies performed using HT-29 cells. The structures of
all potential anti-adhesives in the bovine mixture were confirmed in a fast and sensitive manner by HILIC-HPLC and
offline mass spectrometry (ESI-MS/MS). The methodology proposed here represents a novel approach for the
discovery of anti-adhesive oligosaccharides in diverse feedstocks. Initial screening can be undertaken in hours,
whereas conventional approaches may take days if not weeks. The depletion assay, combined with the HILIC
methodology, facilitates the structural and functional characterization of animal milk oligosaccharides and could be
used to detect any bacteria-oligosaccharide interactions. Indeed, our approach could be exploited for the discovery of
free oligosaccharides with the capacity to bind whole bacterial cells from food sources other than milk.
Jonathan Lane is in receipt of a Teagasc Walsh Fellowship. This work was funded by the Department of Agriculture and Food,
Ireland, under the Food Institutional Research Measure, project reference number 05/R&D/TD/368 and done in
collaboration with the Alimentary Glycoscience Research Cluster (AGRC) Science Foundation Ireland under Grant No.
08/SRC/B1393
43
Sialylation and fucosylation of human milk α1-acid glycoprotein during the first two weeks of
lactation
MAGDALENA ORCZYK-PAWIŁOWICZ1 AND LIDIA HIRNLE2
1Department
of Chemistry and Immunochemistry, Wrocław Medical University, Bujwida 44A, 50-345 Wrocław,
Poland, 2Department of Obstetrics and Gynaecology, Clinic of Reproduction and Obstetrics, Wrocław Medical
University, Dyrekcyjna 5/7, 50-328 Wrocław, Poland
The biological function of α1-acid glycoprotein (AGP), acute phase glycoprotein (40% of sugars), is not clear,
although a number of activities in vitro and in vivo indicate that it may immunomodulate an inflammatory
response and, at the same time, act as a plasma transport protein. Oligosaccharides of AGP are reported to
influence its biological activities, and modulate cell-to-cell interactions and cellular signaling. Under pathological
conditions, AGP may serve as a general protective agent in infections and against toxins by binding to toxic lectin
endotoxins and bacterial lipopolysaccharide. AGP might also play anti-inflammatory and immunomodulatory
roles in inflammation and cancer through its N-glycans, especially its highly branched and α1,3-fucosylated Nglycans. Van Dijk and coworkers [1998] have suggested that AGP molecules expressing sialo-Lewisx
glycodeterminant may interfere with leukocyte-endothelial interactions by binding to E- or P-selectin,
manifesting its anti-inflammatory properties.
The aim of the present work was to study the alterations in relative amounts of sialic acids and fucoses linked by
different anomeric linkages to subterminal N-glycans of human milk AGP in comparison to plasma AGP, and in
relation to stages of human lactation during the first two weeks. The relative amounts of fucosyl- and sialylglycotopes on AGP were analyzed in human milk samples by lectin-ELISA using fucose- (LCA, Lens culinaris, LTA,
Tetragonolobus purpureus and UEA-1,Ulex europaeus) and sialic acid (MAA, Maackia amurensis and SNA,
Sambucus nigra)-specific biotinylated lectins.
We have found that the sialylation and fucosylation patterns of human milk AGP were different than those
reported for human plasma AGP. The observed changes were qualitative and quantitative. The normal plasma
AGP showed very low expression of fucosylated forms. In contrast, the human milk AGP was highly decorated
with fucoses. The fucosylation pattern of normal plasma AGP is limited to the innermost α1,6-fucose, whereas
milk AGP contained higher relative amounts of the innermost α1,6-linked fucose and α1,2-and α1,3-linked
fucoses on outer arms. However, among milk AGPs, no significant differences between samples of milk taken
during the first and the second week of lactation were observed. The sialylation of human milk AGP was higher
than normal plasma AGP for both types, α2,3- and α2,6-, of sialic acid linkages with a predominance of α2,6sialylated form.
We can hypothesize that terminal sugars of milk AGP are biologically active, with anti-inflammatory properties
in modulation of inflammatory processes: the highly sialylated and fucosylated oligosaccharides of milk AGP
could ensure homeostasis during the first weeks of lactation. However, the obtained results are preliminary, they
provide the starting point for further structural and functional studies. In future, it seems to be interesting to
analyze oligosaccharides of human milk AGP in inflammatory diseases, e.g. mastitis.
Funded by: partly by UDA-POKL.04.01.01-00-010/08-01
44
The relative amounts of fucose isoforms in oligosaccharides of human milk fibronectin
MAGDALENA ORCZYK-PAWIŁOWICZ1, LIDIA HIRNLE2 AND IWONA KĄTNIK-PRASTOWSKA1
1Department
of Chemistry and Immunochemistry, Wrocław Medical University, Bujwida 44A, 50-345 Wrocław,
Poland, 2Department of Obstetrics and Gynaecology, Clinic of Reproduction and Obstetrics, Wrocław Medical
University, Dyrekcyjna 5/7, 50-328 Wrocław, Poland
Fibronectin (FN), a multidomain and multifunctional large glycoprotein which is engaged in some cellular
biological functions, e.g. adhesion, migration, proliferation, tissue repair, extracellular matrix remodelling. It is
possible thanks FN multiple domain interactions with its natural ligands, such as fibrin, heparin, collagen, Creactive-protein, and complement components. Moreover, FN serves binding sites to many pathogens through
peptide and/or oligosaccharide sequences, thus being important for bacterial adhesion and colonization to host
tissue.
In human organism FN is an abundant component. FN produced by various cells (e.g. fibroblasts, chondrocytes,
lymphocytes, endothelial cells) is known to be trapped into insoluble multimeric fibrills, while that present in
plasma originate from hepatocyte synthesizes, and is released to blood as a compact globular dimer.
Human FN contains 5-9% of oligosaccharides mainly N- type, and to a lesser degree as O-type. The extent and
type of FN glycosylation varies depending on the tissue source and cell type, and human condition. The plasma
and cellular FNs differ regarding the number of antennae, and the degree of sialylation and α1,6-fucosylation on
the innermost GlcNAc of the chitobiose core. Plasma-derived oligosaccharides can be bi- and tri-antennary,
heavily sialylated and weakly fucosylated. In contrast, cellular FN has bi-antennary glycans, largely fucosylated,
but weakly sialylated. The expression of the α1,2-linked fucosylated glycotope of FN seems to be dependent on
the site of FN synthesis. FN produced by hepatocytes and released into the blood lacks α1,2-linked fucose,
whereas the amniotic and seminal FNs are heavily decorated by α1,2-fucoses.
The N-glycosylation confers FN stability and protects against proteolytic degradation, and also may act as
modulators of FN affinity to some substrates: a lack of oligosaccharides on FN markedly enhanced its ability to
promote adhesion and spreading of fibroblasts, concomitantly increasing affinity to gelatin. The mucin-type Oglycans might play a significant role in segregating the neighboring domains and maintaining the topology and
function of FN domains.
This communication focuses on the analysis of the relative degree of α1,6, α1,3, and α1,2-linked fucoses on FN
present in human milk samples during the first two weeks of lactation of healthy mothers who delivered healthy
newborns. The relative amounts of exposed fucosylated glycotopes were analyzed by lectin-ELISA using LCA
(Lens culinaris), LTA (Tetragonolobus purpureus), and UEA-1 (Ulex europaeus) lectins with well-defined sugar
specificity. The analyses indicate the significant differences in the expositions of fucosylated glycotopes on FN
present in blood plasma and human milk. It was found that the milk FN was heavily decorated with α1,6-, α1,3-,
and α1,2-linked fucoses, whereas the plasma FN negligible. Since the α1,2-fucosylated glycans are known to be
implicated in interaction between host and some pathogens, it can be postulated that α1,2-fucosylated FN in
human milk can act as a natural inhibitor against pathogenic bacteria colonization. The α1,2-fucosylated
glycotopes of soluble milk glycoproteins could constitute one of the element of an innate immune system for
breastfed infants.
45
Effects of specific milk oligosaccharides on the expression of interleukin-8 and marker enzymes
of intestinal cell maturation
KRISTINE ANNA SCHOLAND1, SABINE KUNTZ2, CLEMENS KUNZ2, KLAUS-PETER ZIMMER1 AND SILVIA RUDLOFF1,2
1Department
2Institute
of Pediatrics, University of Giessen, Feulgenstr. 12, D-35392 Giessen, Germany
of Nutritional Science, University of Giessen, Wilhelmstr. 20, D-35392 Giessen, Germany
The microbial colonization of the neonatal gut has a major impact on the development of intestinal functions,
nutrient bioavailability and the immune system. Human milk oligosaccharides (HMO) may not only be substrates
for specific bacterial species, but also resemble their adhesion molecules or ligands which may enable them to
directly affect gut maturation or influence inflammatory processes.
Therefore, we investigated the expression of trehalase (Treh), a disaccharidase regarded as a general marker for
gut maturation, and of fucosyltransferase (Fut) 1 and 2 being involved in the postnatal shift of intestinal surface
glycosylation from sialylated towards more fucosylated structures. An inflammatory response was determined
as interleukin (IL)-8 expression. Human intestinal cells (Caco-2, HT-29) were incubated with 2 mmol of 2’- or 3fucosyllactose (FL), lacto-N-tetraose (LNT), neo-LNT, lacto-N-fucopentaose (LNFP) I or III, 3’- and 6’-sialyllactose
(SL) or disialyl-LNT for 24 h. mRNA expression of Treh, Fut1, Fut2 and IL-8 was determined using real-time RTPCR.
In HT-29 cells, HMO did not affect the expression of Fut1 and Fut2; Treh was merely expressed in these cells
confirming HT-29 as a non-differentiating cell line. In Caco-2 cells, however, Treh expression increased along
with their differentiation over 14 days post confluence. Whereas HMO except for FL showed the tendency to
reduce Treh expression, Fut1 expression in Caco-2 cells seemed slightly upregulated by most HMO tested; Fut2
expression remained unchanged. Stimulation of Caco-2 cells with the proinflammatory cytokine tumor necrosis
factor alpha (TNF-α) itself had no influence on marker enzyme expression. HMO, in particular LNT, LNFP I and III
as well as 3’-SL decreased Treh expression indicating an inhibitory effect of these oligosaccharides on cell
differentiation. Fut1 expression, on the other hand, was slightly enhanced by sialylated HMO in stimulated Caco2 cells; again, there was no effect on Fut2 expression.
With regard to IL-8 expression, disialyl-LNT as well as LNFP III may have the potential to modulate cytokine
production. Whereas IL-8 expression seemed to be decreased in the presence of disialyl-LNT, LNFP III slightly
enhanced IL-8 expression.
These results confirm previous observations [1] indicating that HMO directly modulate intestinal cell
differentiation. In addition, HMO may also have an impact on cytokine production.
[1] Kuntz S, Rudloff, S, Kunz C. (2008) Br J Nutr 99: 462-471
46
In vivo production of fucose-α1, 2-lactose
KATELYN ZAK AND PENG GEORGE WANG
The Ohio State University, Columbus, Ohio, USA
The third largest component of human milk is complex, minimally metabolized oligosaccharides that serve to
protect the infant’s health both by promoting infant intestinal microbiota and warding away harmful pathogens.
The mother’s blood type cause structural variations and change HMO concentrations change lactation. While
HMOs can promote growth of infant intestinal microbiota, their best characterized function is their prebiotic
effects. HMOs have anti-adhesive effects prohibiting pathogens from binding to intestinal epithelial cells as well
as modifying surface glycans that pathogens use to facilitate their entry. Fucosylated oligosaccharides comprise
eighty percent of all HMOs of which 2-linked fucosyloligosaccharides appear the most biologically significant.
The current hurdle in HMO research is the lack of large scale synthesis of HMOs to further test their biological
significance. From clinical trials, fucose-α1, 2-lactose protects against diarrhea significantly better than non-2linked fucosyloligosaccharides. Fucose-α1, 2-lactose may be produced in large scale quantities by recombinant
E. coli which utilize enzymes FKP (fucokinase pyrophosphorylase) and WbsJ (α1, 2-fucoslytransferase) to
enzymatically synthesize fucose-α1, 2-lactose. FKP is a bifunctional enzyme which catalyzes the reaction
between fucose, ATP, and GTP to produce the expensive and hard to obtain GDP-fucose, meanwhile WbsJ is a
known α1, 2-FucT which demonstrates promiscuous acceptor substrate specificity. This method is cost effective
by regenerating the sugar nucleotides required for the synthesis and require only inexpensive, commercially
available, fucose and lactose.
Funded by: NIH R01 HD061935
47
Sialylated galactosides of human milk as inhibitors of enterovirus 71 and A (H1N1) 2009
influenza infections
BETSY YANG1,2, HAU CHUANG2 AND RON-FU CHEN2
1University
of North Carolina, Class of 2014, Chapel Hill, NC 27509, USA
2Department
of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan
Background and specific aims:
Many viruses recognize specific sugar residues, such as sialylated glycosides, as the infection receptors. Human
milk contains many sialylated oligoglycosides which may block viral infections to gastrointestinal tract. We
postulated in the illustration below that sialic acid (SA)-linked alpha 2,3 galactosides (SA-2,3Gal) and SA
2,6Gal from human milk (
) could inhibit infection of
Flu
Sialylated glycans
EV71
enterovirus 71 (EV71) or swine A(H1N1) influenza. We
Intestinal cells
have previously shown that SA-2,6Gal and/or SA-2,3Gal
from human milk specifically inhibited EV71 infection to
DLD-1 intestinal cells (Virol J. 2009 Sep 15; 6:141). This study has been extended to investigate whether SA2,6Gal and/or SA-2,3Gal from human milk inhibited swine A(H1N1) influenza infection to DLD-1 intestinal
cells.
Results:
EV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Pretreatment of DLD-1 cells with
sialidase (2mU, 2 hours) significantly reduced 20-fold EV71 replication (p<0.01). Both SA-2,3Gal and SA2,6Gal from human milk significantly inhibited EV71 infection of DLD-1 cells; SA-2,6Gal from human milk was
more significantly than SA-2,3Gal in inhibiting swine A(H1N1) influenza infection to DLD-1 cells. Depletion of
O-linked glycan or glycolipid, but not N-linked glycan, significantly decreased viral infection of DLD-1 cells,
suggesting SA-linked O-glycans and glycolipids but not N-glycans on DLD-1 cells were responsible for infections
of both viruses.
Summary and implications:
Sialylated galactosides from human milk could significantly inhibit Flu A and EV71 infection of DLD-1 cells,
suggesting sugar-lectin interaction as receptors for viral infection, and natural food with SA-linked glycans can
protect intestinal cells from flu and EV71 infections. Further studies will make a SA-containing glycan linked to
an antivirus agent for making a “double-edge sword” compound to bind EV71 and kill EV71 simultaneously.
48
The oligosaccharide (OS) phenotype of preterm infants predicts risk: A potential indication for
HMOS administration?
ARDYTHE L. MORROW, KURT R. SCHIBLER, JAREEN MEINZEN-DERR, DIANA TAFT AND BARBARA DAVIDSON
Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Ohio,
USA
The human milk oligosaccharides (HMOS) are analogs of oligosaccharides (OS) expressed on infant mucosal
surfaces and in saliva. Understanding the role of HMOs to prevent morbidity and mortality in breastfed infants
also requires understanding the innately expressed OS in the infant. Using banked samples from an extant
cohort, we previously reported that absent or low expression of salivary H antigen (Fuc1,2 oligosaccharide
produced by transferases of the FUT2 gene) – an indirect measure of intestinal expression – significantly predicts
risk of necrotizing enterocolitis or death in preterm infants (J Pediatrics, 2011).
To confirm and extend that finding, we launched a prospective study of infants under 33 weeks’ gestational age
enrolled in three level III neonatal intensive care units (NICUs) in Cincinnati, Ohio (study ongoing). In the first
175 enrolled infants, we analyzed saliva samples at day of life 8 by enzyme-linked immunoassay to determine H
antigen and other oligosaccharide epitopes as predictors of necrotizing enterocolitis or death. FUT2 genotype
was determined independently, and was consistent with salivary phenotype. Using the same cut-point, we again
found that low or absent H antigen significantly predicted subsequent necrotizing enterocolitis or death (odds
ratio =7.7; p=0.004), controlling for low expression of Lewis a (Fuc1,4 oligosaccharide produced by
transferases of the FUT3 gene), infant gestational age and birthweight.
We hypothesize that low or absent H antigen is a biomarker for aberrant intestinal colonization of the preterm
infant. The innate risk that we have identified in relation to low or absent H antigen expression in preterm
infants suggests a prophylactic role for HMOS, which are rich in fucosylated oligosaccharide, and stimulate the
growth of beneficial bacteria. Analysis of the intestinal microbiome and the role of maternal HMOS is underway
in a subset of infants with longitudinal samples.
Sponsored by U.S. National Institute of Child Health and Human Development, HD 13021 and HD 059140
49
Natural antibodies against milk oligosaccharides
MAKSIM NAVAKOUSKI, NADEZHDA SHILOVA, POLINA OBUKHOVA, NAILYA KHASBIULLINA AND NICOLAI BOVIN
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia
Recently anti-glycan antibodies of 106 adult healthy donors’ sera were profiled with the help of printed
glycan array (PGA) which contained about 200 glycans. It was shown that natural human anti-glycan antibodies
bind at least 160 glycans [1], including milk oligosaccharides: LNT (95% of examined donors), LNnT (90%), 2`fucosylLac (90%), Leb-Lac (80%), Lac (75%), 3`SL (60%), 6`SL (35%). The glycans were immobilized on the slide
surface as N-glycine derivatives. In addition, the strong interaction between sera and some glycans closely
related to milk OS was found, e.g. with SiaLeC (99%), LeC (95%), P1 (75%), LeD (50%), asialo-GM1 (100%), GA2
(95%), LeY-Lac (90%).
Anti-milk OS antibodies (anti-milkOS), namely against LNT, LNnT, LeBLac and Lac were detected in
blood of 3-day infants receiving mixed feeding. These anti-milkOS belong to IgG class only and thus seem to be
maternal.
Currently accumulated data don’t allow us to figure out the origin of natural anti-milkOS. On one hand a
reason of their appearance might be natural glycation conjugates of milk OS with proteins due to elevated
concentrations of both components in colostrum; the conjugates could work as immunogens. On the other hand,
the antibodies actually could be directed to different epitopes, in other words, milk OS play a role of mimetics for
unknown yet antigens.
1.
Huflejt, M.E., et al., Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges.
Mol Immunol, 2009. 46(15): p. 3037-49.
Funded by: European Commission’s Marie Curie program (the EuroGlycoArrays ITN)
50
Primary prevention of allergic diseases by probiotics: impact of HMOs
GER T. RIJKERS1,2, TITIA NIERS1, NICOLE RUTTEN2, SASKIA VAN HEMERT3, MAARTEN HOEKSTRA1, ARINE VLIEGER2
1University
Medical Center Utrecht, 2St. Antonius Hospital, Nieuwegein, 3Winclove Bioindustries, Amsterdam, The
Netherlands
Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to
prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by
perinatal supplementation of selected probiotic bacteria. In a double-blind, randomized, placebo-controlled trial,
a mixture of probiotic bacteria selected by in vitro screening (Bifidobacterium bifidum, Bifidobacterium lactis,
and Lactococcus lactis, as Ecologic®Panda) was prenatally administered to mothers of high-risk children
(positive family history of allergic disease) and to their offspring for the first 12 months of life. Probiotics were
administered daily in a dose of 1 x 109 cfu per strain. The placebo-group received an equivalent amount of
carrier material (rice starch plus maltodextran) daily. Up to 80% of mothers breast fed their babies for an
average of 7.1 months. Parental-reported eczema during the first 3 months of life was significantly lower in the
intervention group compared with placebo, 6/50 vs. 15/52 (P=0.035), a relative risk reduction of 58%. The
number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. Similarly, physician-confirmed
eczema during the first 3 months was 3/50 and 11/52 in the intervention and control groups, respectively
(P=0.021). The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis.
In 30% of babies in the placebo group Lc. Lactis was found, which may a consequence of breast feeding. At age 3
months, in vitro production of IL-5 in anti-CD2/CD28 mAb-stimulated whole blood was decreased in the
probiotic group compared with placebo (P=0.04). In conclusion, Ecologic®Panda reduced the incidence of
eczema in high-risk children at 3 months of life, an effect which seems to be sustained throughout the first 2
years. Additional studies at 5-6 years assessing lung function and asthma development are ongoing.
We are planning to study the potential synergistic effect of HMOs on immunomodulation by probiotics. To that
end we will first determine in in vitro studies which HMOs show the highest potential to modulate the neonatal
immune system for induction of Th1 and Treg cells.
Funded by: The Wilhelmina Children's Hospital and the Ministery of Economic Affairs
51
THE SPONSOR
Glycom A/S
www.glycom.com
Glycom is a privately held company founded in 2005 in Copenhagen, Denmark whose focus is to synthesize and
commercialize human milk oligosaccharides (HMOs) along with their precursors and intermediates.
HMOs have been the object of scientific curiosity for over 40 years. Developed over millions of years of human
evolution, HMOs are the 3rd largest component of mother’s milk and are attributed with many of its wonderful
health effects. Until now, study of these natural biopharmaceuticals has been limited and commercialization has
been blocked by lack of available material and high costs – with virtually none available from extractive sources
and only a few synthesized in extremely low quantities and extremely high costs.
Glycom’s breakthrough synthetic chemistry has changed this equation permanently. We have developed efficient
chemical and enzymatic technologies for synthesis of HMOs representing over 50% of total HMO concentration in
typical samples of mother’s milk. The leading HMOs in the Glycom pipeline have established robust scale up
technology in industrial scales and GMP conditions, with glycosylations in multi cubic meter reactors and
production in ton scale and in pure form suitable for human consumption. The opportunity for large scale,
widespread study and commercialization of HMOs has arrived as a result of our efforts and the support of our
partners and investors.
In order to efficiently develop HMOs, Glycom has also developed breakthrough synthesis of the four mono and
disaccharide precursors/building blocks of HMOs. Two of these precursors – sialic acid and L-fucose – are among the
handful of basic natural human sugars. None of them are currently available in sufficient quantities and costs for
high volume commercial applications – including HMO synthesis.
As with the HMOs themselves, the Glycom
technology for the first wave precursors has been scaled up to industrial conditions. As a result, Glycom is already
the world’s largest producer of L-fucose and has already achieved cost levels that are a small fraction of its current
market price. Other precursors are in line for similar breakthroughs.
52
LIST OF PARTICIPANTS
List of Participants
ALBERMANN, CHRISTOPH
AUSTIN, SEAN
Institute of Microbiology, University of Stuttgart, Germany
[email protected]
[email protected]
BEAUPREZ, JOERI
BERING, STINE BRANDT
Ghent University, Belgium
[email protected]
Department of Human Nutrition, Copenhagen University, Denmark
[email protected]
BERTELSEN, HANS
BLANK, DENNIS
Arla Foods, Videbæk, Denmark
[email protected]
Justus-Liebig University, Giessen, Germany
[email protected]
BODE, LARS
BOYLE, ROBERT
University of California, San Diego, USA
[email protected]
Department of Paediatrics Imperial College, London, UK
[email protected]
BØTTKJÆR, KIM
BRASSART, DOMINIQUE
Glycom A/S, Kongens Lyngby, Denmark
[email protected]
Nestec Ltd., Vevey, Switzerland
[email protected]
BØJSTRUP, MARIE
CHEAH, WAI YUEN
Carlsberg Laboratory, Copenhagen, Denmark
[email protected]
Faculty of Science, Macquarie University, Australia
[email protected]
CILIEBORG, MALENE
COPPA, GIOVANNI V.
University of Copenhagen, Denmark
[email protected]
Polytechnic University of Marche, Ancona, Italy
[email protected]
CONTRACTOR, NIKHAT
CRISÀ, ALESSANDRA
Pfizer Nutrition, Collegeville, PA, USA
[email protected]
C.R.A., Monterotondo, Italy
[email protected]
DONOVAN, SHARON M.
DAVIS, STEVEN
University of Illinois, Urbana, USA
[email protected]
Abbott Nutrition, Columbus, OH, USA
[email protected]
DEKANY, GYULA
DOTZ, VIKTORIA
[email protected]
Justus-Liebig University of Giessen, Germany
[email protected]
DROUILLON, MARGRIET
DUNCAN, PETER
University of Ghent, Ghent, Belgium
[email protected]
[email protected]
EL-HAWIET, AMR
ETZOLD, SABRINA
University of Alberta, Canada
[email protected]
Norwich Research Park, Institute of Food Research, Norwich, UK
[email protected]
FERNANDEZ, LEONIDES
FICHOT, MARIE-CLAIRE
Dept. Food Science & Tech, Universidad Complutense de Madrid, Spain
[email protected]
Nestec Ltd., Vevey, Switzerland
[email protected]
FISCHER, LUTZ
FREEZE, HUDSON
University of Hohenheim, Germany
[email protected]
Sanford-Burnham Medical Research Institute, La Jolla, USA
[email protected]
GARRIDO, DANIEL
GEYER, RUDOLF
University of California, Davis, USA
[email protected]
Justus Liebig Universität Giessen, Germany
[email protected]
GRINYER, JASMINE
HALTRICH, DIETMAR
Faculty of Science, Macquarie University, Australia
[email protected]
University of Natural Resources and Life Sciences, Vienna, Austria
[email protected]
HENNET, THIERRY
HESTER, SHELLY
Universität Zürich, Switzerland
[email protected]
[email protected]
53
HINDSGAUL, OLE
HOLSCHER, HANNAH D.
[email protected]
[email protected]
HORLACHER, PETER
IYER, SURI S.
Cognis, Illertissen, Germany
[email protected]
University of Cincinnati, Ohio, USA
[email protected]
JØRGENSEN, JULIE BØCK
KAMERLING, JOHANNIS P.
[email protected]
University of Utrecht, Utrecht, The Netherlands
[email protected]
KATAYAMA, TAKANE
KAVANAUGH, DEVON
Ishikawa Prefectural University, Nonoichi, Japan
[email protected]
Teagasc Food Research Centre, Cork, Ireland
[email protected]
KELM, SØRGE
KITAOKA, MOTOMITSU
University Bremen, Germany
[email protected]
National Food Research Institute, Tsukuba, Japan
[email protected]
KLARENBEEK, BERT
KLASSEN, JOHN S.
Friesland Campina, Beilen, The Netherlands
[email protected]
University of Alberta, Canada
[email protected]
Kovacs, JUDIT
KRENGEL, UTE
[email protected]
Department of Chemistry, University of Oslo, Norway
[email protected]
KRISTENSEN, METTE BACH
KUNZ, CLEMENS
Arla Foods, Viby J., Denmark
[email protected]
Justus Liebig Universität Giessen, Germany
[email protected]
KVISTGAARD, ANNE STAUDT
LANE, JONATHAN
[email protected]
Moorepark Food Research Centre, Cork, Ireland
[email protected]
LEBRILLA, CARLITO
LI, YANQI
[email protected]
Department of Human Nutrition, University of Copenhagen, Denmark
[email protected]
MANURUNG, SARMAULI
MARIOTTA, MARIAROSARIA
Danish Technical University, Veterinary Institute, Denmark
[email protected]
Teagasc Food Research Centre, Cork, Ireland
[email protected]
MAU, SUSANNE
MIKLUS, MICHAEL
[email protected]
PBM Nutritional, Georgia, USA
[email protected]
MILLS, DAVID A.
MORROW, ARDYTHE L.
[email protected]
Cincinnati Children's Hospital, Ohio, USA
[email protected]
MUNBLIT, DANIEL
MUTUNGI, GISELLA
Imperial College, London Clinical Medicine, London, UK
[email protected]
Pfizer Nutrition, Collegeville, PA, USA
[email protected]
MØRKBAK, ANNE LOUISE
NAVAKOUSKI, MAKSIM
[email protected]
Institute of Bioorganic Chemistry, Moscow, Russia
[email protected]
NEWBURG, DAVID S.
NIELSEN, DENNIS S.
Boston College, MA, USA
[email protected]
Department of Food Science, University of Copenhagen, Denmark
[email protected]
ORCZYK-PAWILOXICZ, MAGDALENA
PERONI, DIEGO
Dept. Chemistry & Immunology, Wroclaw Medical University, Poland
[email protected]
Chorus S.p.A., Clinica Pediatrica, Verona, Italy
[email protected]
PETERBAUER, CLEMENS
PRIETO, PEDRO ANTONIO
University of Natural Resources and Life Sciences, Vienna, Austria
[email protected]
Technologico de Monterrey, Mexico
[email protected]
54
PUTZE, JOHANNES
RIJKERS, GER T.
Children's University Hospital Mannheim, Germany
[email protected]
St. Antonius Hospital, Dept. Medical Microbiology, The Netherlands
RÖHRIG, CHRISTOPH H.
SALCEDO DOMÍNGUEZ, JAIME
[email protected]
Facultad de Farmacia, Universidad de Valencia, Spain
[email protected]
SANGILD, PER TORP
SCHOEMAKER, RUUD
[email protected]
Friesland Campina, Deventer, The Netherlands
[email protected]
SCHOLAND, KRISTINE ANNA
SCHWAB, CLARISSA
University Giessen, Germany
[email protected]
University of Vienna, Austria
[email protected]
SKALKAM, MARIA
SOETAERT, WIM
Oxie, Sweden
[email protected]
Ghent University, Belgium
[email protected]
SPRENGER, NORBERT
STAHL, BERND
[email protected]
Danone Research, Friedrichsdorf, Germany
[email protected]
STAUDACHER, ERIKA
TANAKA, HIDENORI
University Vienna, Austria
[email protected]
[email protected]
TAPPENDEN, KELLY
THEROUX, JOHN
[email protected]
[email protected]
THIEM, JOACHIM
THURL, STEPHAN
University of Hamburg, Germany
[email protected]
Fulda University of Applied Sciences, Fulda, Germany
[email protected]
URASHIMA, TADASU
VAN LEUSEN, ELLEN
Obihiro University of Agriculture & Veterinary Medicin, Japan
[email protected]
Friesland Campina, Beilen, The Netherlands
[email protected]
VAN LEEUWEN, SANDER
WALKER, CAREY D.
University of Groningen, Groningen, The Netherlands
[email protected]
Mead Johnson Nutrition, Evansville, IN, USA
[email protected]
WANG, BING
University of Sydney, Australia. Xiamen University & Nestlé Research
Center, Beijing, P. R. China
[email protected]
[email protected]
WEICHERT, STEFAN
Children's University Hospital, Mannheim, Germany
[email protected]
WELMAN, ALAN
WU, SHUAI
Fonterra Coop. Group, Palmerston, New Zealand
[email protected]
University of Davis, CA, USA
[email protected]
YANG, KUENDER D.
YANG, BETSY YAH-HAN
Department of Medical Research, Taiwan
[email protected]
University of North Carolina, Durham, USA
[email protected]
YEH, SHI-HUI
YUE, KE
Chang Gung Institute of Technology, Taiwan
[email protected]
Justus-Liebig University of Giessen, Germany
[email protected]
ZAK, KATELYN
ØSTERGAARD, METTE VIBERG
Ohio State University, Columbus, USA
[email protected]
University of Copenhagen, Department of Human Nutrition, Denmark
[email protected]
55
NOTES
NOTES

Glycobiology of Human Milk Oligosaccharides

Transcription

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