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International Journal of Computational Bioinformatics
and In Silico Modeling
Vol. 2, No. 4 (2013): 152-158
Research Article
Open Access
ISSN: 2320-0634
Modelling transmembrane region in G8 protein of
Human and other organisms
Mamta Sagar1*, Padma Saxena2, Neetesh Pandey1 and Dev Bukhsh Singh3
Department of Bioinformatics, University Institute of Engineering and Technology, Chhatrapati Shahu Ji Maharaj University,
Kanpur-208024
2 Department of Zoology, Dayanand Anglo Vedic Post Graduate College, Chhatrapati Shahu Ji Maharaj University, Kanpur-208024
3 Department of Biotechnology, Institute of Biosciences and Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur208024
1
*Corresponding author: Mamta Sagar; e-mail: [email protected]
Received: 22 May 2013
Accepted: 11 June 2013
Online: 01 July 2013
ABSTRACT
The G8 (for eight conserved glycine residues) domain act as half ABC trasporter is widely distributed, found in
Homo sapiens. Uncharacterized Homolgs of this protein was obtained by sequence similarity search using BLAST
and further analysed by multiple sequence alignment in Pan troglodytes, Nomascus leucogenys, Pongo abelii and
Nomascus leucogenys. Phylogenetic results indicate that G8 protein of Homo sapiens is highly similar to G8
protein of Pan troglodytes, Nomascus leucogenys and closely related. Structure Modelling is done to predict
structural similarities among transmembrane regions and its role as a transporter protein. Transmembrane
prediction shows it is non-cytoplasmic in all organisms. Multiple sequence alignment analysis of transmembrane
region indicates significant similarity except few amino acids, proline at 20th and histidine at 42nd position are
present in human protein but leucine and arginine are present respectively in protein of Pan troglodyte. SSCH(R)
LI motif is predicted among four sequences except in GIR3J8 protein of Nomascus leucogenys. Modeled structure
of G8 proteins of Homo sapiens shows close similarities with its homologues in other three organisms.
Transmembrane region LLWYLVFQYLLPGAGYILR using HMMPLOT is predicted in G1R3J8_NOMLE protein of
Nomascus leucogenys but not in other sequences. Human G8 protein shows similarity to subunit an isoform 1
Q9Z1G4V-type proton ATPase (116 kDa) in Mus musculus which contain V type domain and Vacuolar proton
translocating ATPase protein (Q93050) in Homo sapiens.
Keywords:
G8 protein; ABC half transporter; Modelling; Pan troglodytes; Nomascus leucogenys;
transmembrane
INTRODUCTION
The G8 domain is widely distributed, being found in
proteins from various animals (from Strongylocentrotus
purpuratus to Homo sapiens), lower eukaryotes (such as
Dictyostelium
discoideum
and
Tetrahymena
thermophila) and bacteria (such as the α-proteobacteria
Nitrobacter hamburgensis, γ-proteobacterium Hahella
chejuensis and green non-sulfur bacterium Chloroflexus
aurantiacus) but absent in plants, viruses and archaea.
Many G8-containing proteins are integral membrane
proteins with signal peptides and/or transmembrane
segments, and others lacking TM domain may be
secreted POMGnT1 and TMEM2 proteins, with two
well-conserved glycine residues Guo et al. and the PbH1
http://bioinfo.aizeonpublishers.net/content/2013/4/bioinfo152-158.pdf
domain (SMART: SM00710,Parallel beta-helix repeats
domain) [5 and 9].
G8 protein consists of approximately seventy five (75)
amino acid residues. The secondary structure
prediction of the G8 domain suggests that it contains
ten (10) β-strands and one (01) helix. These strands
are separated by conserved glycine residues and
contain some conserved hydrophobic residues. The G8
domain is actually composed of five β-strand pairs Each
repeat has a sequence resembling hX(0–3)hX(1–
3)GX(1–11)hX(1–3)h, (X represents any amino acid
residues and H represents hydrophobic residues). Most
of the G8-containing proteins are predicted to be
152
Mamta Sagar et. al. / Int J Comput Bioinfo In Silico Model. 2013, 2(4): 152-158
membrane-integral or secretary in nature. The G8
domain may be involved in extra cellular ligand binding
and catalysis. It is an integral membrane receptor
protein with extracellular protein-interaction sites and
intracellular phosphorylation sites and may interact
with extracellular protein–ligands and transduce
intracellular signals to the nucleus [20].
Klein et al. described that G5 and G8 are both halftransporters associated to ABC proteins. These protein
contain of ATPase catalytic domains positioned Nterminal to the transmembrane domain [11].
Venkateswaran et al. (2000) assessed G5 and G8
mediate the efficient excretion of neutral sterols. Lee et
al. predicted ABCG5 (G5) and ABCG8 (G8) involve in
translocation of a diverse substrates across cellular
membranes [6, 8 and 13]. These are ABC transporter
subfamilies in which protein contain one
transmembrane domain one nucleotide binding fold.
These ABC half-transporters make a functional
complex. Sanchez-Fernandez et al. (2001); Yu et al.
(2002) reported that G5 and G8 protein limit the
absorption of sterols and promote cholesterol level. It
has been observed PKHD1 may interact with
extracellular
protein–ligands
and
transduce
intracellular signals to the nucleus [20].
Bergmann et al. detected autosomal recessive
polycystic kidney disease (ARPKD) is associated with
mutations in the polycystic kidney and hepatic disease
(PKHD) gene on chromosome 6p12 [3]. He et al.
predicted a novel protein domain G8 which contains
five repeated β-strand pairs and is present in some
disease-related proteins such as PKHD, KIAA1199,
TMEM (Transmembrane) as well as other
uncharacterized proteins [1&10]. Several other protein
domains frequently co-occur in proteins with a G8
domain. Sabeva et al. reported G5 and G8 proteins are
ABC half-transporters that dimerize within the
endoplasmic reticulum. It mediates in cholesterol
excretion into bile and also noted reduced biliary
cholesterol excretion [17].
Prediction of G8 domain homologues is important for
the research of the structure/function of related
proteins and beneficial for the development of novel
therapeutics. In this study G8 protein sequences is
taken to characterize its function by searching protein
homologs in other organism. Multiple sequence
alignment is done using whole G8 protein and partial
sequences of transmebrane regions predicted from
Phobius tool in Homo sapience Pan troglodytes,
Nomascus leucogenys, Pongo abelii. Modelling was done
to compare transmemrane regions in G8 and its
predicted homologues.
MATERIALS AND METHODS
Homology search and Phyologenetic analysis
BLASTP is used to search homologous sequences [15]
from NCBI using the representative sequences as a
query. Multiple sequence alignment is done for
homologous proein obtained from EBI BLAST to predict
the functions of uncharacterized genes. Phylogram tree
shows how G8 protein of Homo sapiens is more closely
related to proteins of other organisms; Pan troglodytes,
Nomascus leucogenys and Pongo abelii while
cladograms represent relationships between amino
acid sequences.
Transmembrane analysis
The Phobius, a combined transmembrane protein
topology and signal peptide predictor indicates that the
N terminus of the mature protein must be on the noncytoplasmic side of the membrane [14]. The predictor
is based on a hidden Markov model [12]. A model
shows different sequence regions of a signal peptide
and the different regions of a transmembrane protein
in a series of interconnected states.
Homology search with transporter protein using
TCDB BLAST (http://www.tcdb.org/progs/blast.php)
TCDB is a database for transporter protein sequences
and their classification. Blast is performed for G8
protein
of
Human
with
all
proteins
(http://www.tcdb.org/superfamily.php) present in
database [18, 19].
RESULTS AND DISCUSSION
Data base searching, protein sequence pattern analysis,
conserved domain analysis, transmembrane helix
prediction and structure modelling of protein were
done to characterize the features of uncharacterized
proteins.
Data base searching
The G8 protein is highly conserved among the different
organisms such as Pan troglodytes, Nomascus
leucogenys and Pongo abelii indicate good conservation
at the amino acid level (Tables 1 and 2).
G8 protein of Pan Troglodyte’s protein shows
maximum similarity to G8 protein of Homo sapiens
Protein
Multiple sequence alignment of G8 protein and its
homologues obtained by pairwise alignment using
BLAST in different organisms shows that these are
closely related (Figure 1 and 2).Two sequences
TR:H2PIK8_PONAB and TR:G1R3J8_NOMLE showed
only slight variations in G8 protein sequences. In Pongo
abelii (TR:H2PIK8_PONAB) the protein sequence was
92 ٪ similar to G8 protein sequences while in the
Nomascus leucogenys (TR:G1R3J8_NOMLE) the protein
sequence was 83 ٪ similar to G8 protein sequences
(Table -2). The similarity search matches G8 protein
sequences in structure databases (Table - 2), using
different BLAST programs, showed that there were
highly significant (based on E-value and score) closely
related homologues of the G8 protein. It indicates that
Pan troglodyte’s protein show maximum similarity to
G8_HUMAN (Q9UBA6) protein. The results of jalview
indicates that H2QZW7_PANTR Pan troglodyte’s protein
shows difference of two amino acid at position of 45th
& 66th with G8_HUMAN (Q9UBA6) protein and closely
related to human G8 protein as compare to other
153
species. Uncharacterized protein of Pan troglodytes
shows the minimum distance with G8 of human. It is
predicted that both protein shows maximum similarity
(Figure 4).
Table 1. BLAST result for G8 protein of human as a query sequence.
Align
DB:ID
Source
1
SP:G8_HUMAN
Protein
G8
OS=Homo
sapiens
GN=C6orf48 PE=2 SV=2
2
Uncharacterized
TR:H2QZW7_PANTR
troglodytes
protein
Length
Score
Identities
Positives
E
75
414
100.0
100.0
7.0E-39
75
396
97.0
97.0
9.0E-37
75
373
90.0
96.0
4.0E-34
73
353
92.0
93.0
9.0E-32
61
272
83.0
91.0
2.0E-22
OS=Pan
GN=ENSG00000204387
PE=4 SV=1
3
Uncharacterized
TR:G1R3J7_NOMLE
protein
(Fragment)
OS=Nomascus
leucogenys
GN=ENSG00000204387 PE=4 SV=1
4
Uncharacterized
TR:H2PIK8_PONAB
OS=Pongo
abelii
protein
(Fragment)
GN=C6orf48
PE=4
SV=1
5
Uncharacterized
TR:G1R3J8_NOMLE
protein
(Fragment)OS=Nomascus
leucogenys
GN=ENSG00000204387 PE=4 SV=1
Table 2. Similarity score of G8 proteins of Homo sapiens and its homologues in Pan troglodytes, Nomascus leucogenys and Pongo
abelii.
SeqA
Name
Length
SeqB
Name
Length
Score
1
sp|Q9UBA6|G8_HUMAN
75
2
tr|H2QZW7|H2QZW7_PANTR
75
97.0
1
sp|Q9UBA6|G8_HUMAN
75
3
tr|G1R3J7|G1R3J7_NOMLE
75
90.0
1
sp|Q9UBA6|G8_HUMAN
75
4
tr|H2PIK8|H2PIK8_PONAB
73
94.0
1
sp|Q9UBA6|G8_HUMAN
75
5
61
81.0
2
75
3
75
90.0
2
75
4
73
94.0
2
75
5
61
83.0
3
75
4
73
93.0
3
75
5
61
90.0
4
73
5
61
78.0
Figure 1. Jalview of multiple seuqence alignment shows amino acids in different colour, which is used to analyse variation
among; G8 proteins of Homo sapiens and its homologues in Pan troglodytes, Nomascus leucogenys, Pongo abelii and Nomascus
leucogenys.
Figure 2. Representing significant transmembrane region of G8 proteins of Homo sapiens (blue) and its homologues in Pan
troglodyte (green), Nomascus leucogenys (red), Pongo abelii (green). Transmembrane region with high probability and less
probability are shown in red and blue-green color respectively.
154
Multiple sequence alignment
It is confirmed by similarity search result that not only
G8 protein of Homo sapiens is similar to protein
Q9UBA7 but also it is similar to uncharacterized
protein of Pan troglodytes, Pongo abelii and Nomascus
leucogenys, so it is revealed that all proteins are
significantly related to each other and this provide
fundamental basis for characterization of function of
Half ABC transporter protein. No putative conserved
domains have been predicted in these sequences.
Multiple sequence alignment of transmembrane
region
Transmembrane region is predicted in G8 protein of
Homo sapiens, Pan troglodytes, Nomascus leucogenys
and Pongo abelii. Multiple sequence alignment analysis
of this region indicates that there is significant
similarity between different organisms except few
changes, in human protein proline at 20th position and
histidine at 42nd position are present but leucine and
arginine are present respectively in protein of Pan
troglodytes. There are two gaps at the 8th and 29th
position in Pongo abelii while proline and tyrosine are
present on respectively in protein of other organisms.
Nomascus leucogenys (GIR3J8) phenylalanine is present
in place of threonine at 16th position and gap at 35th
position. SSCH(R) LI motif present among four
sequences except in GIR3J8 protein of Nomascus
leucogenys
Figure 3a. Transmembrane prediction in G8 proten of Homo sapiens.
Figure 3b. Transmembrane prediction in G8 like protein of Nomascus leucogenys
155
Figure 4. Phylogenetic tree depict distances among G8 proteins of Homo sapiens and its homologues in Pan troglodytes,
Nomascus leucogenys and Pongo abelii.
Modelling of G8 protein for Tran membrane helix
prediction
Result of present investigation indicates that G8 protein
of Homo sapiens and the likely protein present in other
organisms are non-cytoplasmic (Figure 3a and 3b) and
contain a transmembrane region to be characterized as
a half transporter. Out of five proteins two can be
categorized as ABC transporter protein. This protein
contain significant transmembrane region with high
probability. Mostly G8-containing proteins are
predicted to be membrane-integral or secreted [7]. Its
domain may be involved in extra cellular ligand binding
and catalysis. ABCG8 (G8) is member of the large
family of ATP-binding cassette (ABC) transporters that
facilitate translocation of a wide variety of substrates
across cellular membranes [4 & 16]. The G subfamily is
one ABC transporter protein subfamilies in which
members contain one transmembrane domain and one
nucleotide binding fold. Results of phobius (Figure 3a
and 3b) reveal that functions of homologues proteins
are similar to G8 protein. It is half-transporters and
belonging to the G subfamily of ABC proteins. Based on
the functions of G8-associated domains and proteins, it
is sensible to predict that G8 may be involved in extra
cellular ligand binding and catalysis process.
Transmembrane regions are modeled and positions of
transmembrane and signal peptide region are also
located in modeled structure of G8 protein (Figure 5).
Nucleotide binding domain consists of beta and
extended region of secondary structures which contain
signal peptide region (Figure 5a-5b) Transmembrane
region from amino acid position 48th to 70th and 28th to
60th were predicted in G8 protein of Homo sapiens and
Nomascus leucogenys. Signal peptide is predicted at
start of sequence in Homo sapiens and Pan troglodytes.
Amino acids in G8 like protein of Nomascus leucogenys
vary with G8 protein of Human are found to be located
in transmebrane region.
Figure 5. Transmembrane region (cyan) in G8 protein of (a)
Homo sapience and (b) Nomascus leucogenys and (c) Pan
troglodytes amino acids (orange colour) are highlighted in G8
like protein of Nomascus leucogenys but not present in G8 of
Homo sapiens. Signal peptide is shown in red colour in Pan
troglodytes.
Comparison
of
G8_Human
with
other
Transmembrane (transporter protein)
Transmembrane region was also predicted using
HMMPLOT,
this
predicted
motif
LLWYLVFQYLLPGAGYILR in G1R3J8_NOMLE of
Nomascus leucogenys. Transmembrane region using
HMMPLOT is not predicted in any other sequences.
Human G8 protein shows similarity to subunit a
isoform 1 Q9Z1G4 V-type proton ATPase 116 kDa in
Mus musculus which contain Vtype domain and
Vacuolar proton translocating ATPase protein subunit
(Q93050) in Homo sapiens.
156
The V-type ATPases proteins are related to proton
pumps that acidify intracellular compartments in
eukaryotic cells and have important roles in membrane
trafficking processes. The 116kDa subunit (subunit a)
in the V-type ATPase is involve in proton transport. The
subunit is a transmembrane glycoprotein with
transmembrane helices, hydrophilic amino terminal
and a hydrophobic carboxy terminal. It has roles in
proton transport and assembly of the V-type ATPase
complex encoded by VPH1 and STV1.
These results help to characterize function of ABC half
transporter protein in human and other organism and
predict the amino acid responsible for transportation of
nutrients. These amino acids are VW, D, DS, R, G, S, PS+
L, T, PP which occur individually and in pair in G8
protein of Human. These amino acids are also present
in V-type domain of proton ATPase protein in Mus
musculus and vacuolar proton translocating ATPase
protein subunit (Q93050) in Homo sapiens.
Figure 6. Sequence alignment with V-type proton ATPase subunit of Mus musculus and vacuolar proton translocation ATPase of
Homo sapiens.
CONCLUSION
The G8 domain is widely distributed in both animal and
bacterial proteins including some hereditary disease
related proteins PKHD1, KIAA1109 and TMEM2. Study
provides structural and functional characterization of
this protein. Sequence analysis indicate that G8 protein
of Homo sapiens is closely related to Pan troglodytes,
Nomascus leucogenys and Pongo abelii with similarity
scores of 97, 90, 94 and 81% respectively. Phylogenetic
results reveal that G8 protein of Human is closely
similar to protein H2QZW7_PANTR of Pan troglodytes
and G1R3J7_NOMLE of Nomascus leucogenys.
Transmembrane regions are also similar in G8 protein
of Homo and Pan troglodytes, Although significant
transmembrane region is predicted in G8 protein
Nomascus leucogenys. Results of modelling indicate that
G8 protein of Homo sapiens and predicted homologues
in these organisms are non-cytoplasmic. Structure
modelling of G8 proteins of Homo sapiens revealed
close similarities with its homologues in Pan
troglodytes, Nomascus leucogenys and Pongo abelii,
these
predictions
are
also
supported
by
transmembrane predictions. Signal peptide is also
predicted in G8 protein of human and Pan troglodytes it
is predicted these protein may be derived from same
ancestor.
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© 2013; AIZEON Publishers; All Rights Reserved
This is an Open Access article distributed under the terms of
the Creative Commons Attribution License which permits
unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
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