hereditary hypophosphatemic rickets with

Transcription

Am J Physiol Cell Physiol 302: C1316 –C1330, 2012.
First published December 7, 2012; doi:10.1152/ajpcell.00314.2011.
Processing and stability of type IIc sodium-dependent phosphate cotransporter
mutations in patients with hereditary hypophosphatemic rickets with
hypercalciuria
Sakiko Haito-Sugino,1* Mikiko Ito,2* Akiko Ohi,1 Yuji Shiozaki,1 Natsumi Kangawa,1
Takashi Nishiyama,1 Fumito Aranami,1 Shohei Sasaki,1 Ayaka Mori,1 Shinsuke Kido,1 Sawako Tatsumi,1
Hiroko Segawa,1 and Ken-ichi Miyamoto1
1
Department of Molecular Nutrition, University of Tokushima Graduate School, Tokushima, Japan; and 2University of Hyogo
School of Human Science and Environment, Hyogo, Japan
Submitted 22 August 2011; accepted in final form 6 December 2011
hypophosphate; SLC34A3; misfolding; trafficking; degradation
Na⫹-dependent inorganic phosphate (NaPi) cotransporter
in the brush-border membranes of proximal tubular cells mediates the rate-limiting step in the overall inorganic phosphate
(Pi)-reabsorptive process (3, 19). The type II NaPi cotransporters (SLC34A1/NPT2a/NaPi-IIa, SLC34A3/NPT2c/NaPi-IIc)
are expressed in the apical membranes of proximal tubular
cells and mediate Pi transport (21, 28). The SLC34 family
comprises the electrogenic NaPi-IIa/b and the electroneutral
NaPi-IIc, which display Na⫹:Pi cotransport stoichiometries of
3:1 and 2:1, respectively (7). NaPi-IIa and NaPi-IIc respond to
THE
* S. Haito-Sugino and M. Ito contributed equally to this work.
Address for reprint requests and other correspondence: K. Miyamoto, Dept.
of Molecular Nutrition, Institute of Health Biosciences, Univ. of Tokushima
Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan (e-mail:
[email protected]).
C1316
variations in dietary Pi, parathyroid hormone (PTH), fibroblast
growth factor-23, and other factors by changing their abundance in the apical membranes of proximal tubular cells (3, 19,
21, 27). In rats and mice, NaPi-IIa has the most important role
in renal Pi reabsorption (being responsible for 60 –70%), while
NaPi-IIc is thought to have the most important regulatory role
in weaning animals (1, 22, 28). In adult animals maintained on
a normal Pi diet, however, NaPi-IIc mediates a small percentage of Pi reabsorption (1, 22, 28).
Hereditary hypophosphatemic rickets with hypercalciuria
(HHRH), an autosomal recessive disorder first identified in a
large Bedouin tribe, is characterized by hypophosphatemia
secondary to renal Pi wasting, resulting in increased serum
1,25-dihydroxyvitamin D3 concentrations with associated intestinal Ca2⫹ hyperabsorption, hypercalciuria, rickets, and osteomalacia (34). Clinical studies suggest that HHRH is a
primary renal Pi wasting disorder because all of the associated
abnormalities, with the exception of the reduced renal reabsorption of Pi, can be corrected by dietary Pi supplementation
(34). Initially, the NPT2a gene was thought to be responsible
for HHRH; however, recent studies identified several mutations in the NPT2c transporter gene (SLC34A3) as the cause of
HHRH (2, 10, 14 –17, 23, 24, 33, 43). The fact that HHRH is
caused by NPT2c loss-of-function mutations is compatible
with the HHRH phenotype and the prevailing view of renal Pi
regulation (14).
In a previous study, we showed that mice homozygous for
the disrupted Npt2c gene (Npt2c⫺/⫺) are viable, fertile, and do
not display growth retardation (30). Npt2c knockout (KO)
mice exhibit hypercalcemia and hypercalciuria, but not hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia (30). In contrast, Npt2a KO mice show
hypophosphatemia and hyperphosphaturia in addition to hypercalcemia and hypercalciuria, but not rickets or osteomalacia
(1). To clarify the relative importance of Npt2a and Npt2c, we
studied Npt2a⫺/⫺Npt2c⫹/⫹, Npt2a⫹/⫺Npt2c⫺/⫺, and Npt2a⫺/⫺
Npt2c⫺/⫺ double knockout (DKO) mice (29). Npt2a⫺/⫺
Npt2c⫺/⫺ DKO mice exhibited lower body weight, severe
hypophosphatemia, hypercalciuria, and renal calcification (29).
This phenotype is similar to that seen in HHRH, which is
characterized by hypophosphatemia, short stature, and rickets
with secondary absorptive hypercalciuria (29). A high-Pi diet
can reverse the rickets/osteomalacia in HHRH patients, and a
high-Pi diet after weaning restored plasma Pi levels and rescued the bone phenotypes in Npt2a⫺/⫺Npt2c⫺/⫺ DKO mice
(29). This study showed that, in mice, Npt2a and Npt2c play
0363-6143/12 Copyright © 2012 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on November 2, 2016
Haito-Sugino S, Ito M, Ohi A, Shiozaki Y, Kangawa N,
Nishiyama T, Aranami F, Sasaki S, Mori A, Kido S, Tatsumi S,
Segawa H, Miyamoto K. Processing and stability of type IIc sodiumdependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria. Am J Physiol Cell
Physiol 302: C1316 –C1330, 2012. First published December 7, 2012;
doi:10.1152/ajpcell.00314.2011.—Mutations in the apically located
Na⫹-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPiIIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several
HHRH mutations on the processing and stability of human NaPi-IIc.
Mutations S138F, G196R, R468W, R564C, and c.228delC in human
NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this
reduction is a result of a decrease in the Vmax for Pi, but not the Km.
G196R, R468W, and c.228delC mutants were not localized to oocyte
membranes. In opossum kidney (OK) cells, cell surface labeling,
microscopic confocal imaging, and pulse-chase experiments showed
that G196R and R468W mutations resulted in an absence of cell
surface expression owing to endoplasmic reticulum (ER) retention.
G196R and R468W mutants could be partially stabilized by low
temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in
an aggregation complex. In contrast, S138F and R564C mutants were
trafficked to the cell surface, but more rapidly degraded than WT
protein. The c.228delC mutant did not affect endogenous NaPi uptake
in OK cells. Thus, G196R and R468W mutations cause ER retention,
while S138F and R564C mutations stimulate degradation of human
NaPi-IIc in renal epithelial cells. Together, these data suggest that the
NaPi-IIc mutants in HHRH show defective processing and stability.
MUTATION OF NaPi-IIc IN HHRH
MATERIALS AND METHODS
Plasmid constructs. Full-length wild-type (WT) human NaPi-IIc (aa
1–599) and NaPi-IIa (aa 1– 639) tagged with enhanced green fluorescence protein (EGFP) at the NH2 terminus (pEGFP-C1 vector; Stratagene, La Jolla, CA) (EGFP-NaPi-IIc WT, or -NaPi-IIa WT) were
generated using standard cloning techniques (11). PCR and a site-directed
mutagenesis kit (QuikChange Lightning site-directed mutagenesis kit,
Stratagene) were used to introduce the following mutations into EGFPNaPi-IIc WT: c.228delC, S138F, G196R, R468W, and R564C. Primer
sequences for all mutants are shown below. NaPi-IIc WT and mutant
cDNAs were subcloned into pCMV-Tag2A (Stratagene) (FLAG-NaPiIIc) for Western blotting and Pi uptake assays. All constructs were
confirmed by the following sequencing: c.228delC, 5=-CAGCGTCCTCAAGGCTGCGGGCTCCTCGGC-3= (sense) and 5=-GCCGAGGAGCCCGCAGCCTTGAGGACGCTG-3= (antisense); S138F, 5=AGAGTTCCAGCACGTTCTCCTCCATCGTGGT-3= (sense) and 5=ACCACGATGGAGGAGAACGTGCTGGAACTCT-3= (antisense);
G196R, 5=-GGCTCGGCGGTGCACAGGATCTTCAACTGGC-3=
(sense) and 5=-GCCAGTTGAAGATCCTGTGCACCGCCGAGCC-3=
(antisense); R468W, 5=-CTGGTGCCTGCACTGTGGCTGCCCATCCCGC-3= (sense) and 5=-GCGGGATGGGCAGCCACAGTGCAGGCACCAG-3= (antisense); and R564C, 5=-CTGGAGCCCTGGGACTGCCTGGTGACCCGCT-3= (sense) and 5=-AGCGGGTCACCAGGCAGTCCCAGGGCTCCAG-3= (antisense).
Cell culture and transfection. Opossum kidney (OK) cells were a
gift from Dr. J. Biber (Zurich University, Zurich, Switzerland) and
maintained in appropriate medium (11). For living cell imaging, 4 ⫻
105 cells/dish were plated in 35-mm glass-bottom dishes (MatTek,
Ashland, MA), and subconfluent cultures were transfected with each
EGFP-fused construct. Transfections were performed for 24 h using
LipofectAMINE2000 (Invitrogen, Carlsbad, CA) in accordance with
the manufacturer’s instructions. For immunostaining, 0.5 ⫻ 105 cells/
well were plated on glass coverslips (Matsunami-glass, Osaka, Japan;
15 mm) in 12-well dishes, and subconfluent cultures were transfected
with each EGFP-fused construct. For Pi uptake studies, 0.5 ⫻ 105
cells/well were plated in 12-well dishes and subconfluent cultures
were transfected with each FLAG-fused construct. For Western blotting analysis, 1.4 ⫻ 105 cells/well were plated in six-well dishes, and
subconfluent cultures were transfected with each EGFP or FLAGfused construct.
Pi uptake and protein expression in Xenopus oocytes. WT and
mutant human NaPi-IIc clones were linearized by digestion with XbaI
and transcribed into human NaPi-IIc complementary RNA (cRNA)
using T7 RNA polymerase as previously described (28). Expression
of proteins in Xenopus oocytes and Pi uptake measurements were
performed as described previously (28).
Pi uptake studies in OK cells. Pi transport was studied in monolayers of OK cells transfected with FLAG-fused constructs in 12-well
dishes. Uptake experiments were performed as previously described
(12). In brief, Pi uptake studies were carried out in uptake solution
(containing 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM
MgSO4, 10 mM HEPES-Tris pH 7.4, 0.1 mM KH2PO4/K2HPO4, and
1 ␮Ci/ml 32P; PerkinElmer, Bridgeport, CT) at 37°C. After 6 min, the
cells were washed three times with cold stop solution (containing 137
mM NaCl, 10 mM Tris·HCl pH 7.2, 2 mM KH2PO4/K2HPO4 pH 7.4)
and then solubilized by the addition of 0.4 ml of 0.1 N NaOH.
Aquasol-2 (Packard Instruments, Meriden, CT) was added to cell
lysates. 32P in cell lysates was measured by liquid scintillation
counting. The protein concentration in lysates was determined using a
bicinchoninic acid assay (BCA) protein assay kit (Pierce, Rockford,
IL). Pi transport was calculated as nmoles 32P per mg protein taken up
in 6 min. These experiments were performed in triplicate and repeated
two to four times.
Confocal microscopy analysis. Cells were imaged using a TCS-SL
laser-scanning microscope (Leica Wetzlar, Germany) equipped with a
⫻63 oil immersion objective. Immunostaining was performed as
described previously (12). For protein detection, cells were incubated
with anti-GM130 monoclonal antibody (BD Transduction Laboratories, San Diego, CA; 1:100) or anti-ERp29 polyclonal antibody (Gene
Tex, San Antonio, TX; 1:500). Alexa Fluor 568-phalloidin (Molecular
Probes, Eugene, OR; 1:100) was used for detection of actin. Alexa
Fluor 568-conjugated mouse IgG (Molecular Probes, 1:100) and
Alexa Fluor 568-conjugated rabbit IgG (Molecular Probes, 1:100)
were used as secondary antibodies. Coverslips were mounted with
Aqua PolyMount (Polysciences, Warrington, PA).
Preparation of total cell lysates. Isolation of proteins was performed as previously described (12). OK cells were rinsed twice with
ice-cold Tris-buffered saline (TBS), scraped off in the isolation buffer
(0.5% NP-40, 1 mM PMSF, 2 ␮g/ml aprotinin, and 2 ␮g/ml leupeptin
in TBS) and resuspended five times using a 20-gauge needle. Homogenates were centrifuged at 10,000 g for 2 min, and supernatants were
collected in new tubes. The protein concentration in lysates was
determined using a BCA protein assay kit and analyzed by Western
blotting.
Preparation of oocyte membranes. Oocyte membranes were prepared using a method modified from Tucker et al. (36). In brief, 30 –50
oocytes were suspended in 1 ml of phosphate-buffered saline (PBS)
and homogenized using a 26-gauge needle. Homogenates were centrifuged at 1,000 g for 10 min at 4°C until all yolk granules and
melanosomes were pelleted. Final supernatants were pelleted at
165,000 g for 30 min to generate a total membrane fraction devoid of
yolk granules. This membrane pellet was resuspended in 40 ␮l of
AJP-Cell Physiol • doi:10.1152/ajpcell.00314.2011 • www.ajpcell.org
independent roles in the regulation of plasma Pi and bone
mineralization. Loss of function of NPT2c in humans causes
HHRH, while loss of both Npt2a and Npt2c is required to
cause an HHRH-like phenotype in mice.
It remains unknown how human NPT2c mutation causes
defective Pi transport activity. Homozygous, compound
heterozygous or heterozygous mutations in NPT2c have been
reported in HHRH patients (2, 10, 14 –17, 23, 24, 33, 43).
Frame shift, intronic deletions, adjacent splice sites, and nonsense mutations are predicted to produce abnormal proteins. In
previous studies based on cysteine scanning mutagenesis and
in vitro translation assays, a topology model was proposed for
NaPi-IIa that is most likely also valid for NaPi-IIb and NaPi-IIc
(25). This model predicts 12 transmembrane spanning domains, with intracellular NH2 and COOH termini. Most mutations identified to date are in highly conserved residues in
NPT2c and may be in regions important for function and
cellular trafficking. Recently, Jaureguiberry et al. (14) investigated the functions of T137M and V466Stop human
NaPi-IIc mutants and showed that the T137M mutation
affected NaPi cotransport activity and that the V466Stop
mutation affected transport to the membrane. Generally,
most new protein is synthesized in the endoplasmic reticulum (ER), where it is core-glycosylated at its N-glycosylation sites and folded correctly (39). Upon proper folding and
passing through the ER control system, proteins then undergo further modification before being trafficked to their
final destinations through the secretory pathway. Defective
proteins do not pass the ER quality control system during
processing; these proteins are retained in the ER in misfolded form before undergoing degradation by cytosolic
proteasomes (39). In the present study, the function and
cellular localization of human NaPi-IIc bearing the mutations c.228delC, S138F, G196R, R468W, and R564C were
investigated in renal proximal epithelial cells [opossum
kidney (OK) cells] and Xenopus oocytes.
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C1318
Tris·HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
(vol/vol) Triton X-100] containing a mixture of protease inhibitors (1
mM PMSF, protease inhibitor cocktail; Invitrogen). After 15 min at
⫺80°C, thawed samples were centrifuged for 5 min at 4°C and
supernatants were used for immunoprecipitation. Immunoprecipitation was performed with anti-FLAG M2 monoclonal antibody overnight at 4°C followed by affinity purification using protein G-agarose
beads (Santa Cruz Biotechnology, Santa Cruz, CA) After incubation
for 1 h at 4°C, immunoprecipitates were washed three times in TNE
buffer. Protein samples were subjected to SDS-PAGE and Western
blot analysis and visualized by autoradiography. The band density was
quantified using the ImageJ program (National Institutes of Health,
Bethesda, MD) and is expressed as relative intensity to that of total
NaPi-IIc at time 0.
Determination of half-life and degradation pathway. OK cells were
transiently transfected with FLAG-NaPi-IIc (WT, S138F, G196R,
R468W, or R564C), and experiments using various inhibitors were
performed as described previously (20). After 24 h, the medium was
replaced with a medium containing the new protein synthesis inhibitor
cycloheximide (10 ␮g/ml, Sigma), the proteasome inhibitors MG132
(10 ␮M, Sigma) and lactacystin (10 ␮M, Sigma), or the lysosome
inhibitors chloroquine (100 ␮g/ml, Sigma) and leupeptin (100 ␮g/ml,
Sigma) and incubated for the indicated period of time. After incubation, total cell lysates were prepared and subjected to SDS-PAGE and
immunoblot analysis. The band density was quantified using the
ImageJ program, normalized against that of actin and calculated so
that the normalized density at time 0 was 100% for the half-life study.
The log10 of the percentage of normalized band density is expressed
versus time, and the half-life was calculated from the log10 of 50% for
the protein. For studies of the degradation pathway, the band density
is expressed as relative intensity to that of the control (nontreatment)
level.
Low-temperature treatment. OK cells were transiently transfected
with EGFP-NaPi-IIc (WT, S138F, G196R, R468W, or R564C) and
low-temperature experiments were performed as described previously
(42). After 24 h, cells were incubated at 37°C (control) or 30°C for 24
h in a humidified atmosphere containing 5% CO2. After incubation,
cells were visualized by confocal microscopy or were lysed and
analyzed by Western blotting. The band density was normalized
against that of actin and is expressed as a percentage of the total
NaPi-IIc level present in the mature form. These experiments were
performed in duplicate and repeated two to four times.
Blue native-polyacrylamide gel electrophoresis. NaPi-4 (opossum
endogenous NaPi-IIa; oNaPi-IIa) polyclonal antibodies were obtained as described previously (12). A peptide identical to the
carboxyl-terminal 12-amino acids (LGVLSQHNATRL) of opossum NaPi-IIa (oNaPi-IIa, NaPi-4), with a cysteine at the NH2
terminus for conjugation to keyhole limpet hemocyanin, was
generated and used to generate rabbit antibodies (Sigma Genosys,
Hokkaido, Japan). Affinity purification of anti-oNaPi-IIa antibodies was carried out by elution from a column to which the antigen
had been coupled (12). A blue native-polyacrylamide gel electrophoresis (BN-PAGE) system (NativePAGE Novex Bis-Tris Gel
System) was purchased from Invitrogen and used according to the
manufacturer’s protocols and as previously described with some
modification (4, 32). Isolated crude membrane fractions were pelleted
by centrifugation at 17,000 g for 1 h and were dissolved in the sample
buffer supplied with the system containing 3% n-dodecyl-␤-D-maltoside (DDM). The lysate protein concentration was determined using a
BCA protein assay kit. Protein complexes were separated using
4 –16% gradient bis-Tris gels (Invitrogen) at 4°C. Gels were subjected
to Western blot analysis. Protein samples were diluted to a final
concentration of approximately 1 g/ml in sample buffer containing 3%
DDM and 0.75% G-250 sample additive (supplied with the system).
Into each lane of a native gel, 2.5 ␮g of protein were loaded. The
NativeMark Unstained Protein Standard (Invitrogen) was used as a
molecular weight marker. Gels were run and blotted to PVDF mem-
PBS, and samples were subjected to SDS-polyacrylamide gel electrophoresis.
Immunoblot analysis. Cells grown on six-well dishes were transfected as described above and previously (12). In brief, equivalent
amounts of protein samples were heated at 95°C for 3 min in sample
buffer containing 5% 2-mercaptoethanol, subjected to 8 or 10%
SDS-PAGE, and transferred electrophoretically to polyvinylidene
difluoride (PVDF) transfer membranes (Immobilon-P; Millipore, Billerica, MA). Membranes were incubated in 5% skim milk in 20 mM
Tris·HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 (TBST) at
room temperature for 1 h to inhibit nonspecific binding, and then
overnight at 4°C with anti-FLAG M2 monoclonal (Sigma, St. Louis,
MO; 1:2,000), anti-EGFP monoclonal (Clontech, Palo Alto, CA;
1:5,000), or anti-actin monoclonal antibody (Chemicon, Temecula,
CA; 1:5,000; used as an internal control), in 1% skim milk in
TBST, followed by treatment with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove,
PA). Signals were detected using the Immobilon Western system
(Millipore).
Cell surface biotinylation. Biotinylation was performed using the
Pierce cell surface protein isolation kit (Pierce) according to the
manufacturer’s instructions and as previously reported with some
modifications (12). OK cells grown in six-well dishes were rinsed
three times with ice-cold PBS. The cells were then incubated with
2 ml of biotin solution containing 0.25 mg/ml sulfo-NHS-SS-biotin
(EZ-Link Sulfo-NHS-SS-Biotin, Pierce) for 30 min at 4°C, with
gentle shaking on an orbital shaker. The quenching solution supplied in the kit was added to the cells. Labeled cells were scraped
off gently and transferred to tubes. The cells were collected at 500
g for 3 min at 4°C and washed twice with ice-cold TBS. Cells were
lysed in 0.2 ml of a lysis buffer (supplied in the kit) containing
protease inhibitors for 1 h on ice, and homogenized by repeated
pipetting. The lysates were cleared at 10,000 g for 2 min at 4°C,
and the supernatants were transferred to new 1.5-ml tubes. Biotinylated proteins were precipitated overnight with avidin-agarose
beads (Immobilized NeutrAvidin Gel, Pierce) at 4°C. The beads
were collected at 1,500 rpm for 3 min at 4°C, washed three times
in 1 ml of wash buffer (supplied in the kit), then resuspended in
100 ␮l of sample buffer containing 50 mM DTT, incubated at 95°C
for 5 min, and centrifuged for 3 min at 1,500 rpm. Supernatants (15
␮l) were subjected to SDS-PAGE and blotted with anti-FLAG M2
monoclonal antibody as described above.
Analysis of glycosylation and brefeldin A treatment. Cell lysates
prepared from OK cells transfected with FLAG-NaPi-IIc (WT,
S138F, G196R, R468W, or R564C) were digested with 1 mU of
glycopeptidase F (also called peptide:N-glycosidase F; PNGase F,
TaKaRa, Shiga, Japan) for 15 h at 37°C under denaturing conditions
with the incubation buffer as recommended by the manufacturer’s
protocol (38). Digestion with endoglycosidase H (Endo H, New
England BioLabs, Ipswich, MA) was performed for 1 h at 37°C
according to the manufacturer’s protocol (37). After incubation with
sample buffer for a further 3 min at 95°C, samples were subjected to
SDS-PAGE and immunoblot analysis. Brefeldin A (BFA) treatment
was performed as previously described with some modifications (26).
In brief, the medium was replaced with a medium containing BFA (10
␮g/ml, Sigma) at 15 h after transfection and incubated for 24 h at
37°C before samples were collected.
Metabolic labeling and immunoprecipitation. Metabolic labeling
was performed as described previously with some modifications (44).
After 24 h, cells were transiently transfected with FLAG-NaPi-IIc and
starved of methionine in cysteine- and methionine-free Dulbecco’s
modified Eagle’s medium (Invitrogen) for 1 h at 37°C. Starvation
medium was removed and replaced with medium containing [35S]methionine/cysteine (EXPRESS Protein Labeling Mix, [35S]⫺,
PerkinElmer). After 40 min, cells were washed twice with PBS and
lysed immediately or replaced with normal medium for the chase time
periods. Collected cells were suspended in TNE lysis buffer [20 mM
C1319
branes using the XCell II Blot Module (Invitrogen). Briefly, the PVDF
membrane was prewet for 30 s in methanol. All pads were soaked in
1⫻ Transfer Buffer (Invitrogen) to remove air bubbles and arranged
in a standard stack of pads and filter paper. After transfer, PVDF
membranes were incubated in 8% acetic acid for 15 min to fix the
proteins, then rinsed with deionized water and air dried. Membranes
were then rewetted with methanol to remove any background dye
bound to the membranes and rinsed with deionized water before
blocking. PVDF membranes were blocked with TBST containing 5%
skim milk for 60 min before overnight incubation with antibodies
diluted in 1% skim milk in TBST. FLAG-NaPi-IIc and NaPi-4 were
detected by anti-FLAG M2 monoclonal antibody and anti-NaPi-4
affinity purified antibody as described above.
Statistics. Statistical significance (P ⬍ 0.05) of differences between
means was determined using paired or unpaired t-tests.
Mutations of human NaPi-IIc transporter with HHRH.
Figure 1 shows the positions of the human NaPi-IIc mutations detected in HHRH patients. Human NaPi-IIc has four
predicted N-glycosylation consensus sites (N-X-S/T) within
a large third extracellular loop. The S138F, G196R, and
R468W mutations are localized in the first, second, and last
intracellular loops of the transporter. The R564C mutant is
located in the COOH-terminal region of the transporter. The
c.228delC deletion causes a shift in the open reading frame
after codon 76, which is predicted to result in the translation
of a novel sequence of 74 aa that is unrelated to NaPi-IIc
and is then followed by a termination codon at codon 151.
The predicted protein lacks all 12 membrane-spanning domains as well as the COOH-terminal intracellular tail of
NaPi-IIc.
Functional analysis of human NaPi-IIc mutants in Xenopus
oocytes and OK cells. In Xenopus oocytes expressing c.228delC,
S138F, G196R, R468W, or R564C mutants, NaPi transport
activities were determined and compared with that in cells
expressing WT transporter (Fig. 2A). To compare 32Pi uptake
among oocytes injected with different clones, we ensured that
all groups were injected with 25 ng of cRNA/oocyte. The
cRNA concentration of all clones was measured by absorption
at 260 nm and also by resolving cRNA in 1% agarose/
formaldehyde gels. Pi ion transport kinetics observed in human
WT NaPi-IIc were very similar to those previously observed in
Fig. 1. Structure of human Na⫹-dependent phosphate
(NaPi) cotransporter NaPi-IIc and scheme showing
hereditary hypophosphatemic rickets with hypercalciuria (HHRH)-associated mutations. Based on a topological structure of human NaPi-IIc, the locations of
amino acid substitutions (shaded circles) associated
with HHRH mutations are shown as reported by Radanovic et al. (25). The HHRH mutations used in the
present study are indicated in the boxes. Four putative
N-glycosylation sites are indicated by asterisks (*).
RESULTS
mouse and human NaPi-IIc (28). As shown in Fig. 2B, 32Pi
uptake in all clones exhibited saturation curves that are compatible with Michaelis-Menten behavior. In WT protein and
S138F and R564C mutants, a plateau was reached with an
extracellular Pi concentration ⬍500 ␮M, and we observed no
significant differences in Km values (Km ⫽ 60 – 80 ␮M). In
other mutants, we could not determine Km values exactly
because of very low activities. In contrast, the Vmax value of
each mutant was significantly decreased compared with that of
WT (WT ⫽ 4.5 pmol/min per oocyte, c.228delC ⫽ 0.4, S138F ⫽
1.1, G196R ⫽ 0.4, R468W ⫽ 0.6, R564C ⫽ 1.8). In addition,
we also determined the amounts of mutant proteins in the
oocyte membrane (Fig. 2C). The amounts of S138F and
R564C mutant proteins were slightly reduced compared
with the amount of WT transporter. The c.228delC, G196R,
and R468W mutants were not detected in our assay. Immunohistochemical analysis showed weaker fluorescence signals in the membranes of oocytes expressing S138F or
R564C mutants compared with those expressing WT protein, while no signals were detected in those expressing
G196R and R468W mutants (Fig. 2D). Thus, all mutants
exhibited a significant reduction in surface expression compared with WT NaPi-IIc, with a profile similar to that
observed in functional expression analysis.
We also analyzed transport activity in OK cells transfected
with the NaPi-IIc gene (Fig. 2E). NaPi transport activity was
increased about fivefold in WT NaPi-IIc-expressing cells compared with endogenous Pi uptake (cells transfected with empty
vector). Transport activity was massively decreased in OK
cells expressing c.228delC, S138F, G196R, R468W mutants,
the activities of which were almost identical to that of the mock
controls. The transport activity of the R564C mutant was about
half that of WT NaPi-IIc in OK cells. Thus, the transport
activities of all mutants tested in this experiment were markedly reduced.
Membrane localization of human NaPi-IIc transporter in
OK cells. Using confocal microscopy, we observed cellular
localization of EGFP-NaPi-IIc WT protein and mutants in OK
cells (Fig. 3A). As shown in a previous study using EGFPfused mouse NaPi-IIc, we observed that the NaPi-IIc WT
protein was exclusively localized at the cell surface and within
apical patches in OK cells (12). S138F and R564C mutants
C1320
were also clearly detected in the apical membranes of OK cells.
The c.228delC mutant transporter was mainly localized to
cellular components and not detected in the apical membrane.
In addition, G196R and R468W mutants were mainly detected
in intracellular compartments in OK cells, but not the apical
membrane.
Next, we investigated the expression of WT and mutant
proteins by Western blot analysis. As shown in Fig. 3B, we
performed Western blot analysis using FLAG-NaPi-IIc. The
FLAG-NaPi-IIc protein was detected as bands of ⬃65 kDa and
⬃130 kDa. S138F and R564C mutants were detected as mature
protein (⬃130 kDa); however, G196R and R468W mutants
were detected only as the lower band (⬃65 kDa). No protein
bands were detected in lysates from cells expressing FLAGfused c.228delC mutant transporter (data not shown).
Fig. 2. Functional analysis of NaPi-IIc mutations. A: oocytes injected with water, cRNA for human NaPi-IIc wild-type (WT) protein, or cRNAs for each mutant
protein were assayed after 2 days for uptake of Pi (100 ␮M) in 96 mM NaCl medium (n ⫽ 8 experiments). Values are means ⫾ SE. B: Pi concentration
dependence of NaPi-IIc-mediated Pi uptake. NaPi-IIc-mediated Pi uptake was measured at 3, 10, 30, 100, 300, and 1,000 ␮M Pi in standard uptake solution and
is plotted against the Pi concentration. The Pi uptake was saturable and fit to the Michaelis-Menten curve. Values are means ⫾ SE (n ⫽ 6 experiments).
C: Western blot analysis of human NaPi-IIc in Xenopus oocytes. The expression of mutant transporters (WT, c.228delC, S138F, G196R, R468W, or R564C)
in Xenopus oocyte lysates was analyzed by Western blotting. D: expression of human NaPi-IIc mutants in oocyte membranes. The localization of human NaPi-IIc
in Xenopus oocytes was analyzed as described in MATERIALS AND METHODS. E: Pi uptake assays were performed in opossum kidney (OK) cells transiently
expressing FLAG empty vector (vec), FLAG-NaPi-IIc (WT, c.228delC, S138F, G196R, R468W, or R564C), or FLAG-NaPi-IIa. Results are expressed as
means ⫾ SE (n ⫽ 3) of uptake values obtained from multiple inserts from at least two independent experiments. The dotted line indicates baseline endogenous
activity (empty vector). aP ⬍ 0.05 vs. empty vector. bP ⬍ 0.05 vs. NaPi-IIc WT.
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As shown in Fig. 3C, we performed a biotinylation protein
assay in OK cells expressing FLAG-NaPi-IIc WT and mutants.
Cell surface labeling in OK cells was performed as described
in MATERIALS AND METHODS. The results indicated that the
biotinylated cell surface proteins were mature protein (⬃130
kDa) and that the smaller proteins (65 kDa) are unlikely to be
localized at the cell surface. The results also indicated that the
G196R and R468W mutants are not localized at the cell
surface. In contrast, mature protein was detected in cells
expressing WT protein and those expressing S138F and R564C
mutants.
ER localization of human NaPi-IIc mutant. Next, we examined the cellular localization of EGFP-NaPi-IIc mutants by
immunostaining (Fig. 4). Immunocytochemistry analysis revealed that WT NaPi-IIc staining was exclusively detected in
the apical membrane patches (colocalization with actin). In
contrast, G196R and R468W mutant proteins displayed an
immunofluorescence staining pattern that was more restricted
to a perinuclear ER-like distribution. In addition, most of the
immunoreactivity for WT and mutant NaPi-IIc was not detected in the Golgi apparatus. Indeed, these mutants did not
show any colocalization with cell-surface marker protein, indicating that G196R and R468W mutants were not expressed at
the cell surface. These data strongly suggest that the lack of
transport activity associated with G196R and R468W mutants
resulted from the absence of the protein from the cell surface.
Effects of glycosidase on human NaPi-IIc mutant. As shown
in Fig. 5, we compared the glycosylation state of FLAG-NaPiIIc proteins by assessing their sensitivities to two different
glycosidases: Endo H, an enzyme that hydrolyzes the high
mannose and hybrid-type N-glycans present on immature secretory proteins in the ER, but not complex forms of Nglycans, and PNGase F, an enzyme that cleaves nearly all
N-glycans. Because conversion of high mannose or hybrid
glycans into complex glycans occurs in the Golgi, resistance to
Endo H indicates that a glycoprotein has reached this compartment. On the contrary, Endo H sensitivity is considered to be
an indicator of immaturity. The protein size of FLAG-NaPi-IIc
WT transporter after treatment with PNGase F was the smaller
of the two bands detected (Fig. 5A). Treatment with PNGase F
resulted in a shift in molecular mass to ⬃60 kDa, indicating
that NaPi-IIc contains N-glycans weighing ⬃65 kDa. Thus, the
upper band (⬃130 kDa: band a) seems to be a mature glycosylated form, and the lower band (⬃65 kDa: band b) seems to
be an immature core-glycosylated form, while the two bands of
⬃60 kDa observed following PNGase F treatment seem to be
a partially digested form and a nonglycosylated form (bands c
and d). Treatment of S138F and R564C mutants with PNGase
F resulted in a similar pattern to that for WT protein (from
band b to bands c and d). Treatment of G196R and R468W
mutants with PNGase F shifted the molecular weight similar to
WT protein (from band b to bands c and d), suggesting that
Fig. 3. Expression of human NaPi-IIc in OK cells. A: OK cells were transfected with enhanced green fluorescent protein (EGFP)-NaPi-IIc (WT, c.228delC,
S138F, G196R, R468W, or R564C) and then processed for confocal microscopy. The xz cross section is indicated in the bottom panels. B: cells were transfected
with FLAG-NaPi-IIc (WT, S138F, G196R, R468W, or R564C), separated by SDS-PAGE, and subjected to immunoblot analysis using antibodies against FLAG.
Left: short exposure. Right: long exposure. C: surface biotin labeling of OK cells expressing FLAG empty vector, FLAG-NaPi-IIc WT, or mutants. At 48 h after
transfection, the biotin-labeled proteins were examined as described in MATERIALS AND METHODS. The mutant proteins were run on more than one gel.
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they only reach an immature core-glycosylated form. We did
not investigate the difference between bands c and d in the
present study.
As shown in Fig. 5B, treatment with Endo H demonstrated
that the lower single FLAG-NaPi-IIc band (65 kDa) was Endo
H sensitive, confirming that it was retained in the ER as an
immature protein. The ⬃130 kDa band of WT NaPi-IIc was
Endo H resistant. Similarly, the smaller bands (⬃65 kDa) of
S138F, G196R, R468W, and R564C mutants were sensitive to
Endo H. These data also show that the G196R and R468W
mutants were located in the ER. In addition, the mature bands
(⬃130 kDa) of S138F and R564C were Endo H resistant like
WT protein. These observations suggest that S138F and
R564C mutants can be trafficked to the cellular membrane.
Effect of brefeldin A on the ER export of human NaPi-IIc
mutant. Using BFA, a compound that blocks protein trafficking
from the ER to the Golgi, we investigated whether the defect in
trafficking from the ER causes an increment of immature
bands. As shown in Fig. 6A, we investigated the effect of BFA
on the amounts of FLAG-NaPi-IIc (immature form, ⬃65 kDa).
After BFA treatment, the amounts of NaPi-IIc WT, S138F and
R564C mutant protein (⬃65 kDa) were significantly increased
compared with those in nontreated control cells (Fig. 6B). In
contrast, those of G196R and R468W mutants were not different. These data indicate that G196R and R468W mutants
remain in the ER.
Human NaPi-IIc mutations impair maturation of the
transporter. To further determine the kinetic effects of maturation of WT protein and G196R and R468W mutants or
degradation, we performed a pulse-chase experiment. As observed above, WT NaPi-IIc after the pulse appeared as a single
band of ⬃65 kDa, which was then progressively converted to
a more slowly migrating band (around ⬃130 kDa), representing the mature NaPi-IIc protein. As illustrated in Fig. 7,
although both mutants were synthesized as the ⬃65-kDa immature form at a level similar to that of WT NaPi-IIc, no
mature band was detected at any of the indicated time intervals
during the chase. This led to an overall ER retention of the
G196R and R468W mutants due to prevention of maturation
relative to WT NaPi-IIc. This absence of maturation of G196R
and R468W mutants was also observed by Western blot analysis (Fig. 3, B and C).
Effects of protease inhibitors on maturation of human NaPiIIc mutants. On the basis of the above findings, the absence of
the mature and functional forms of G196R and R468W resulted from ER retention and degradation. Accordingly, treatment with proteasome and lysosome inhibitors might provide
important insights into possible mechanisms of mutant NaPiIIc trafficking and degradation. Cells were treated with MG132
or chloroquine, and their lysates were subjected to Western
blot analysis. As shown in Fig. 8, A and C, exposure to MG132
significantly increased the protein levels of the mature forms
(⬃130 kDa) of FLAG-NaPi-IIc WT with an apparent increase
Fig. 4. Localization of human NaPi-IIc mutants in cells. OK cells transfected with EGFP-NaPi-IIc (WT, c.228delC, S138F, G196R, R468W, or R564C) were
stained with antibodies against phalloidin (actin marker), GM130 (Golgi marker), and ERp29 [endoplasmic reticulum (ER) marker] and then processed for
confocal microscopy. EGFP-NaPi-IIc is shown in green, the intracellular marker is shown in red, and the overlay of both signals is shown in yellow. The xz cross
section is indicated in the bottom panels.
in the levels of their immature forms (⬃65 kDa). Similarly,
the amounts of immature bands of S138F, G196R, R468W,
and R564C were significantly increased after MG132 treatment. The amounts of the mature bands (⬃130 kDa) of
S138F mutant were also increased (approximately twofold)
after treatment with MG132, as were those of WT. We did
not detect the mature forms of G196R and R468W mutants
after MG132 treatment.
In contrast, lysosomal inhibition by chloroquine had no
action on the immature forms of the WT, G196R and R468W
proteins (Fig. 8, B and D). These results provide additional
evidence that the G196R and R468W mutants are largely
trapped in the ER. In contrast, lysosomal inhibitors markedly
increased the levels of the mature bands (⬃130 kDa) of S138F
and R564C mutants. The same results were obtained using
leupeptin (data not shown). Thus, degradation of R564C mutants involves the lysosome degradation pathway and that of
S138F mutant involves both proteasome and lysosome pathways.
Half-lives of mutant NaPi-IIc transporters. Next, we determined the half-lives of the mutant transporters in OK cells
(Fig. 9). FLAG-NaPi-IIc WT and mutants were expressed in
OK cells, and the amounts of mature mutant protein bands
(FLAG-fused) were analyzed after treatment of cells with
cycloheximide. The mature form (⬃130 kDa) of WT protein
exhibited a half-life of 9.5 h. The S138F and R564C mutants
were degraded more rapidly (half-life ⫽ 4.0 h and 3.0 h,
respectively). The half-lives of the immature form (⬃65 kDa)
of WT and S138F and R564C mutant proteins were 0.7 h, 1.77
h, and 0.1 h, respectively. The half-life of the immature form
of S138F mutant was thus about twice that of WT protein. The
half-lives of G196R and R468W mutants (immature form)
were even more extended to 15.3 h and 9.64 h, respectively.
These data suggest that the S138F and R564C mutants are
unstable in the apical membrane compared with WT protein. In
addition, the S138F mutant (immature form half-life ⫽ 1.77 h)
is retained in the ER for longer than WT protein (half-life ⫽
0.7 h). We also determined the half-lives of EGFP-NaPi-IIc
mutants. The mature forms of the S138F and R564C mutants
were more rapidly degraded than that of the WT protein (data
not shown).
Stability of G196R and R468W mutant at 30°C. As shown in
Fig. 7, G196R and R468W mutant proteins are not able to be
converted to the mature glycosylated form over time during
biosynthesis. We therefore asked whether conditions such as
low temperature could stabilize the G196R and R468W mutants, because this condition is favorable for protein folding
and has been shown to stabilize misfolded mutant protein (6).
OK cells expressing EGFP-fused G196R or R468W were
incubated at a low temperature (30°C) (Fig. 10A). These cells
showed a higher percentage (⬃10% and 20%, respectively) of
the mature glycosylated form (⬃150 kDa) of G196R and
R468W than OK cells incubated at 37°C (Fig. 10C). In contrast, the amounts of the mature form (⬃150 kDa) of WT or
S138F or R564C mutants were not affected in the low temperature experiment (Fig. 10, A and B). These data indicate that
a low temperature favors protein folding of G196R and R468W
mutants.
Oligomeric state of NaPi-IIc. Electrophoretic separation by
BN-PAGE facilitates subsequent SDS-PAGE and immunoblotting, which permitted us to demonstrate the presence of
a protein complex. In a previous study, the combination of
BN-PAGE, immunoprecipitation, and LC-MS/MS analysis
Fig. 6. Effect of brefeldin A (BFA) on the trafficking of human NaPi-IIc.
FLAG-NaPi-IIc was expressed in OK cells. At 15 h after transfection, the
culture medium was replaced with medium with or without BFA and incubated
for a further 24 h. Cells were lysed using each appropriate preparation and
separated by SDS-PAGE (A) or imaging data (B). A: FLAG-NaPi-IIc was
detected using an anti-FLAG M2 antibody. The mutant proteins were run on
more than one gel. B: the band density was measured and normalized to that
for actin, and then compared with bands from untreated (⫺) cells and treated
with BFA (⫹). Each value represents the mean ⫾ SE of 3–5 independent
experiments performed in duplicate.
Fig. 5. Glycosidase sensitivity of human NaPi-IIc protein expressed in OK
cells. A: the cell lysates of OK cells transfected with FLAG-NaPi-IIc (WT,
S138F, G196R, R468W, or R564C) were digested with peptide:N-glycosidase
F (PNGase F). The lysates were separated by SDS-PAGE, and FLAG-NaPi-IIc
was detected by immunoblotting using an antibody against FLAG. Bands a, b,
c, and d indicate the predicted mature form (a), immature form (b), and
deglycosylated form (c and d), respectively. The mutant proteins were run on
more than one gel as indicated by the spaces between samples. B: total cell
lysates of OK cells expressing FLAG-NaPi-IIc were digested with endoglycosidase H (Endo H). Proteins were separated by SDS-PAGE and probed with
anti-FLAG M2 antibody.
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C1324
demonstrated the presence of NaPi-IIa protein complex in
the brush-border membrane of renal epithelial cells (32). In
the present study, BN-PAGE was used to assess the oligomeric states of NaPi-IIc mutants in OK cells (Fig. 11A). We
also investigated whether the quaternary structure of NaPiIIc is important for ER exit. EGFP-NaPi-IIc and FLAGNaPi-IIc proteins were detected by Western blot analysis.
Under our experimental conditions, we suggest that human
NaPi-IIc may form an oligomeric state. The NaPi-IIc complex appeared as two predominant bands with apparent
molecular mass of about 250 and 500 kDa when analyzed by
BN-PAGE. The 250- and 500-kDa bands may represent
dimers and tetramers of the NaPi-IIc transporter. A weaker
additional band of ⬃720 kDa was observed with WT NaPiIIc. The oligomeric state of R564C mutant protein in BNPAGE was similar to that of WT protein. In contrast, G196R
and R468W mutants showed slow mobility and high molecular weights, suggesting that G196R and R468W mutants
may be denatured or present in an aggregation complex. The
oligomeric state of the S138F mutant was similar to that of
G196R and R468W mutants. We suggest that the G196R
and R468W mutants failed to fold properly in the ER and
that folding of the S138F mutant may also be affected, but
that it can exit from the ER to the Golgi.
Furthermore, we investigated whether the expression of
WT and mutant NaPi-IIc proteins affected the conformation
of endogenous NaPi-IIa (NaPi-4) in OK cells (Fig. 11B). In
the lysates of OK cells expressing NaPi-IIc WT or mutant
proteins, a prominent band of ⬃280 kDa was observed
following BN-PAGE, suggesting that NaPi-4 protein is
present as a dimer. Coexpression of each mutant protein did
not affect the oligomeric state of NaPi-4 in OK cells. In
addition, following SDS-PAGE, we analyzed the levels of
NaPi-4 protein in OK cells expressing each mutant. The data
show that NaPi-4 protein (⬃130 kDa) levels were not
different between OK cells expressing WT and those expressing mutant NaPi-IIc (Fig. 11, C and D). In addition, we
assessed Na⫹-dependent Pi cotransport activity in OK cells
expressing WT and mutant NaPi-IIc. The suppression of
endogenous Na⫹-dependent Pi transport was not observed in
this experiment (Fig. 2E). No mutant transporters
(c.228delC, S138F, G196R, R468W, and R564C) had dominant negative effects on downregulation of endogenous
NaPi-4 protein in OK cells by functional analysis and
Western blot analysis.
DISCUSSION
In the present study, we made five mutants of human
NaPi-IIc detected in HHRH patients and investigated the
pathophysiology of these NaPi-IIc mutations. Ghezzi et al. (8)
suggested that electrogenic and electroneutral NaPi-II isoforms
probably share a common functional architecture, which includes substrate coordination sites and their translocation pathway. These structural features are reflected in the identical
transport characteristics (similar Km for Pi, Km for Na, pH
dependent) for these isoforms. In contrast, the mechanisms of
maturation/trafficking and degradation for NaPi-IIc remain
unknown.
G196R and R468W mutants. Heterozygosity for one of
the missense mutations in the other kindreds was associated
with increased calcium excretion and other laboratory abnormalities, such as hypophosphatemia and elevated serum
1,25(OH)2D levels; these mutations included G196R and
R468W. The patient with compound heterozygosity of
G196R and R468W had nephrocalcinosis and hypophosphatemia due to renal Pi wasting, hypercalciuria, and elevated 1,25(OH)2D levels. In addition, Kremke et al. (15)
Fig. 7. Pulse-chase analysis of human NaPi-IIc protein. As described in MATERIALS AND METHODS, OK cells were labeled with [35S]methionine/cysteine
for 1 h and chased with unlabeled methionine/cysteine for the indicated times. FLAG-NaPi-IIc was then immunoprecipitated with anti-FLAG M2
antibody, separated by SDS-PAGE, and visualized by autoradiography (top). The amount of FLAG-NaPi-IIc at each time point was quantified using the
ImageJ program (National Institutes of Health, Bethesda, MD) and is indicated as relative intensity to that of total FLAG-NaPi-IIc at 0 h (bottom).
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also reported that renal calcification was found in homozygous carriers of G196R in NaPi-IIc. G196R and R468W do
not seem to pass the ER quality control system. Indeed,
Endo H and PNGase F treatment showed that G196R and
R468W mutants are expressed as immature proteins and
retained in the ER. Pulse-chase experiments showed that
complex glycosylation appears to be important for cell
surface delivery and membrane retention of NaPi-IIc. The
mutant proteins (G196R and R468W) were not detected as
mature glycosylated forms. In addition, when the cells were
incubated at 30°C, partial rescue of the G196R and R468W
NaPi-IIc proteins out of the ER to the apical membrane was
evident. In BN-PAGE analysis, we showed that NaPi-IIc
WT protein might exist as a dimer or tetramer in nonreduc-
ing conditions, but G196R and R468W mutants were not
detected as dimers and may be aggregated. The protein
misfolding may be caused by the G196R and R468W
mutations. These data suggest that the conformation of
NaPi-IIc in the ER may be essential for ER exit. Taken
together, these studies suggest that the G196 and R468 in
the NaPi-IIc protein may be essential for proper folding in
ER. Moreover, we suggest that the G196R and R468W
mutations are unlikely to affect endogenous NaPi-4 complexing and function (Fig. 2E and Fig. 11C).
HHRH is an autosomal recessive disease, and biallelic
mutations are required from complete manifestation of the
phenotype. However, previous studies of HHRH kindreds
have indicated that some subjects with a single mutation
Fig. 8. Effect of protease inhibitors on the expression of human NaPi-IIc mutants. OK cells were
transiently transfected with FLAG-NaPi-IIc (WT,
S138F, G196R, R468W, or R564C). After 24 h,
cells were cultured in the absence (⫺) or presence
(⫹) of MG132 (10 ␮M; A) or chloroquine (100
␮g/ml; B) and the cells were analyzed at 24 h. Cell
lysates were separated by SDS-PAGE and subjected
to Western blot analysis using the anti-FLAG
antibody. Actin was used as an internal control. C
and D: band density was normalized against that
of actin and is expressed as relative intensity to
that of the control (nontreatment) level. Chlo,
chloroquine. Values are means ⫾ SE. Data were
taken from A and B.
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(i.e., carriers) can show clinical features of HHRH, including hypercalciuria and osteopenia (2, 16). We investigated
whether NaPi-IIc mutants had an influence on the structure
of NaPi-IIc WT (data not shown). In consequence, we did
not observe dimmer formation between WT and mutants in
OK cells. However, we must examine the final conclusion
about these using other methods in more detail.
Export of NaPi-II family members from the ER is poorly
understood. Such ER export requires specific signals in the
amino acid sequence and posttranslational modifications that
Fig. 10. Effect of low temperature on the
expression of human NaPi-IIc mutants in OK
cells. A: OK cells were transfected with
EGFP-NaPi-IIc (WT, S138F, G196R or
R468W). Cells were transfected with each
plasmid and then incubated at 37°C (control)
or 30°C for 24 h. EGFP-NaPi-IIc was visualized by fluorescence microscopy. The xz
cross section is indicated in the bottom panels. B: the lysates of OK cells under these
conditions were separated by SDS-PAGE and
subjected to immunoblot analysis using an
antibody against EGFP. The mutant proteins
were run on more than one gel. C: imaging
analysis of the mature form of the WT protein
and each mutant protein following SDSPAGE. Actin was used as an internal control.
Values were calculated from band densities
of the mature form divided by the sum of
density in bands for the mature and immature
forms, multiplied by 100. Means ⫾ SE from
two to four independent experiments performed in duplicate are shown.
Fig. 9. Biochemical half-life of NaPi-IIc protein. OK cells were transiently transfected
with FLAG-NaPi-IIc (WT, S138F, G196R,
R468W, or R564C). After 24 h, cells were
further cultured in the presence of cycloheximide and lysed at the indicated times. Cell
lysates were subjected to Western blot analysis using the antibody to FLAG. Actin was
used as an internal control. The amount of
FLAG-NaPi-IIc at each time point was quantified using the NIH ImageJ program, normalized against that of actin, and Iis indicated as a percentage of FLAG-NaPi-IIc at 0
h, which was set as 100%. The log10 of the
percentage normalized band density is expressed vs. time (T), and the half-life (T1/2)
was calculated from the log10 of 50% level
for the mature or immature band of FLAGNaPi-IIc. Each data point represents the
mean ⫾ SE of duplicate determinations.
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probably include glycosylation and oligomerization (39). Posttranslational processing in the ER is critical for the proper
trafficking of proteins to their cellular targets, including the
plasma membrane, secretory vesicles, and other organelles.
The ER is the major site where newly synthesized membrane
proteins such as NaPi-IIc enter the secretory pathway and
become folded (39). Only proteins that are correctly folded and
assembled are usually able to leave the ER (18). Most misfolded or unassembled proteins are retained in the ER and
subsequently degraded in the cytosol by a process known as
ER-associated protein degradation (18). In addition, the ER
stress response plays a role in a number of pathophysiological
processes, including pancreatic beta cell apoptosis and kidney
disease (5, 31). The residue R468 is located in the last intracellular loop and highly conserved in the NaPi-II transporter
family. In a previous study, we demonstrated that PEX19 binds
to a dibasic motif (K/R-K motif) in the last intracellular loop of
NaPi-IIa, and that this motif appears to be important for
NaPi-IIa internalization by PTH (11). In addition, Iwaki et al.
(13) reported that the missense mutation A499V of mouse
NaPi-IIa, within the last intracellular loop, might lead to
protein misfolding. In the present study, we suggest that the
mutant proteins (G196R and R468W) are retained in the ER
and may cause ER stress in renal epithelial cells (Fig. 12).
S138F mutant. In a previous study, a female diagnosed
with HHRH was reported as having one maternally inherited
mutation (S138F) and one paternally inherited mutation
(S192L) (14). In the present study, we have not addressed
the function of S192L, which is located in the adjacent
transmembrane region of human NaPi-IIc. Furthermore,
Jaureguiberry et al. (14) showed that the mutation of T137
to methionine (M) may lead to an uncoupling of cotransport
and that this uncoupling may render cotransport, and thus
the rate of Pi uptake, less efficient. Surface expression of
T137M was only reduced to 40% of the WT level and did
not seem to fully explain this lack of function. This mutation
did not affect Kd, but it decreased Vmax of the mutant
transporter for Pi. Therefore, we focused on the S138F
mutation of the human NaPi-IIc transporter.
The S138F mutation occurs in residues that are conserved
in members of the type II NaPi transporter gene family.
S138 is located in one of two conserved repeat regions in
NaPi-IIa family members, and it is likely to be involved in
transport functions, making it an important residue for
proper folding of the protein (7, 14, 41). In the experiment
using Xenopus oocytes, NaPi cotransport activities were
markedly decreased in the S138F mutant compared with
those of WT protein. In terms of the cell surface expression
Fig. 11. Blue native (BN)-PAGE analysis of the oligomeric structure of human NaPi-IIc and standard SDS-PAGE analysis. A: cell lysates were purified in
nondenaturing buffer with 3% n-dodecyl-␤-D-maltoside extracts of OK cells, resolved by BN-PAGE, and analyzed by Western blotting. Black arrowheads
indicate the WT pattern. B: effect of NaPi-IIc expression on the oligomeric state of endogenous NaPi-4. NaPi-4 signal is shown by the arrow. C: SDS-PAGE
analysis of NaPi-IIc and NaPi-4. The same membrane was reprobed with anti-NaPi-4 antibodies. Shown are short exposure (top left) and long exposure (top right)
of WT, c.228delC, and S138F. D: densitometric analysis of NaPi-4 bands. Data are expressed as a percentage of the WT level ⫾ SE. These experiments were
performed two to four times.
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of S138F mutant in oocytes, the mutant protein was clearly
localized in the cell membrane, suggesting that the S138F
mutation causes functional loss of NaPi transport activity in
oocytes. In addition, we examined the production and cellular trafficking of the S138F mutant NaPi-IIc protein in OK
cells. A mature complex of the S138F mutant, like WT
NaPi-IIc protein, is resistant to Endo H digestion and
expressed at the cell surface, as assessed by cell surface
immunofluorescence and biotinylation. In contrast, the immature bands of S138F mutant are more stable than those of
the WT protein, suggesting that the S138F mutant is partially prevented from exiting the ER. The degradation of
S138F mutant was more rapid than that of WT protein, and
proteasome and lysosomal inhibitors increased the amounts
of S138F protein. Thus, in addition to its role in NaPi
transport activity, the S138 residue has an important role in
intracellular trafficking and membrane stability (Fig. 12).
R564C mutant. In a previous study, we identified the
R564C mutation in Japanese HHRH patients (43). In the
present study, WT and R564C human NaPi-IIc were Nglycosylated in the ER and then further modified upon
appropriate folding and passed to the ER quality control
system before apical expression. The levels of R564C mutant protein were markedly increased in OK cells treated
with lysosome inhibitors. Membrane proteins are frequently degraded in lysosomes, but there are some examples of transporter
proteins being degraded by the ubiquitin-proteasome system. In a previous study, we showed that the WLHSL motif
in the COOH-terminal region of mouse NaPi-IIc is essential
for localization to the apical membrane (12). In addition,
recent studies demonstrated that the COOH-terminal region
of NaPi-IIc is associated with PDZK1 in the apical mem-
ACKNOWLEDGMENTS
We thank Dr. Y. Kanai (Osaka University Graduate School of Medicine,
Osaka, Japan) and Dr. N. Anzai (Kyorin University School of Medicine,
Tokyo, Japan) for thoughtful comments and advice.
GRANTS
This study was supported by a grant from Grant-in Aid for Scientific
Research on the Priority Area “Transportsome” (17081013) and by Grants
18590891, 20590975 (to M. Ito), 18390250, and 20390236 (to K. Miyamoto)
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan. This work was also supported by a Grant Culture of Japan and the
Human Nutritional Science on Stress Control from the 21st Century Center of
Excellence Program.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the
author(s).
AUTHOR CONTRIBUTIONS
S.H.-S., M.I., H.S., and K.-I.M. conception and design of the research;
S.H.-S., M.I., A.O., Y.S., N.K., T.N., F.A., S.S., A.M., S.K., S.T., and H.S.
Fig. 12. Mechanisms underlying cellular trafficking of human NaPi-IIc WT
and mutant proteins in OK cells. Mature NaPi-IIc WT protein is processed and
trafficked to the apical membrane. The NaPi-IIc mutants G196R and R468W
are retained in the ER as misfolded proteins. S138F and R564C were processed
and trafficked to the apical membrane but were rapidly degraded by the
proteasome and/or lysosomes.
brane and, in PDZK1 null mice, this protein is important for
adaptation to a low Pi diet (9, 40). We have no information
on the interaction of the R564C mutant and PDZK1, but the
mutant is able to undergo trafficking to the apical membrane
in OK cells, suggesting that the WLHSL motif of the
transporter is not influenced in the R564C mutant. However,
this mutation may be involved in interactions with binding
proteins. Thus, the COOH-terminal region of human NaPiIIc appears to be essential for apical membrane insertion and
stability.
c.228delC mutant. A homozygous c.228delC mutation was
initially classified as being responsible for idiopathic hypercalciuria (34), while several members of a Bedouin kindred
shown to be heterozygous for the c.228delC mutation displayed mild hypophosphatemia, reduced tubular maximal Pi
reabsorption/glomerular filtration rate (TmP/GFR), and elevation in 1,25(OH)2D levels in addition to increased urinary calcium excretion (35). However, loss of one NaPi-IIc
allele does not always lead to laboratory abnormalities (2).
In the Bedouin kindred, heterozygosity for the c.228delC
mutation did not always lead to hypercalciuria, which indicates that additional factors contribute to the presence or
absence of these urinary changes. In the present study, the
c.228delC deletion was predicted to result in a complete loss
of function of the NaPi transporter. Using analyses of NaPi
transport activity, Western blotting, and immunohistochemical analyses of OK cells expressing c.228delC mutant, the
mutant proteins showed no dominant negative effect on the
expression of endogenous NaPi-IIa transporter (NaPi-4)
(Fig. 2E and Fig. 11C).
Finally, we showed that human NaPi-IIc WT is N-glycosylated in the ER and then undergoes further modification upon
appropriate folding and passing the ER quality control system,
which is followed by apical expression. The protein misfolding
may be caused by the G196R and R468W mutations, while
S138F and R564C mutants may be unstable in renal epithelial
cells. These results provide further information about the
cellular regulation (synthesis and degradation) of the human
NaPi-IIc transporter. The pathogenesis of HHRH is still poorly
understood and further research into other mutations associated
with HHRH is required.
performed the experiments; S.H.-S., M.I., S.T., H.S., and K.-I.M. analyzed
data; S.H.-S., M.I., H.S., and K.-I.M. prepared the figures; S.H.-S., M.I., H.S.,
and K.-I.M. drafted the manuscript; M.I., H.S., and K.-I.M. edited and revised
the manuscript; H.S. and K.-I.M. approved the final version of the manuscript.
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