Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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The
electrogenic Na+-HCO
transporter; pH measurement; acid-base mechanism
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INTRODUCTION |
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THE MOST IMPORTANT pH
buffer system in the body is CO2/HCO
The key step in understanding the molecular physiology of electrogenic
Na+-HCO
The primary structure of rat NBCe1-A predicts a protein of 1,035 amino acids that is 30-40% identical to the AEs. Topological studies on AE1 suggest that the protein has cytoplasmic NH2 and COOH termini and as many as 14 transmembrane (TM) segments (14, 32). AE1 is N-glycosylated once in the fourth extracellular loop, between TM7 and TM8, whereas AE2 and AE3 have consensus glycosylation sites in the third extracellular loop, between TM5 and TM6. The oligosaccharide on human AE1 takes two forms, a long-chain polylactosaminyl structure or a shorter complex structure (24). Mutation of the N-glycosylation site in AE1 reduces targeting the protein to the cell surface in Xenopus oocyte studies, but the nonglycosylated protein is still functional (19). One role of N-glycosylation in AE1 may be to permit the interaction of AE1 with calnexin, an endoplasmic reticulum chaperone; altering the N-glycosylation site by mutagenesis eliminates the interaction of AE1 with calnexin (31). AE2 is also glycosylated, but it lacks sialic acids (44). The glycosylation status of AE3 is unknown.
The deduced amino acid sequence predicts that rat NBCe1 has seven putative consensus sites for N-linked glycosylation. On the basis of an AE1 topology model (14), three of the sites (N33, N199, and N208) are on the cytoplasmic NH2 terminus, one (N497) is within TM3, and the remaining three (N592, N597, and N617) are on the third extracellular loop. These last three sites are thus candidates for glycosylation. Western blots show that NBCe1 migrates at 130 kDa in the kidney (38), whereas the deduced amino acid sequence predicts a 116-kDa protein. Thus at least one of the consensus sites may be glycosylated. In the present study, we investigated the oligosaccharide composition of NBCe1-A using a glycosidase enzyme assay and a lectin-binding assay. To locate the N-linked glycosylation sites, we individually mutated asparagine (N) to glutamine (Q) at all seven putative glycosylation sites and also created double and triple mutants. We analyzed these mutants by expressing them in Xenopus oocytes and examining the molecular weight (MW) of each mutant transporter after treatment of peptide N-glycosidase F (PNGase F). We also performed microelectrode experiments to assess the function of mutant transporters. We found that NBCe1-A is normally glycosylated at two sites on the third extracellular loop and that the unglycosylated protein has normal function.
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MATERIALS AND METHODS |
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Extraction of crude membranes. A whole kidney from adult Sprague-Dawley rats or rabbits was removed under anesthesia (intraperitoneal injection, 100 mg/kg pentobarbital sodium). A frozen kidney from a cow was obtained commercially, and a kidney from the Ambistoma tigrium salamander was isolated as described previously (38). Slices of the kidney from each species were placed in an ice-cold homogenization buffer (HB)-I (250 mM sucrose, 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin) and homogenized using a Polytron. For extraction of crude plasma membranes of oocytes expressing NBCe1, oocytes were homogenized in hypotonic HB-II (7.5 mM sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin) as described previously (10). Homogenates were centrifuged at 750 g in a microcentrifuge (Eppendorf, model 5415C) for 5 min at 4°C to remove cell debris; the supernatant was centrifuged for 30 min at 16,000 g at 4°C. The pellet, which contained most plasma and organellar membranes, was used for analysis.
Deglycosylation.
Membrane fractions (200 µg) isolated from rat tissues were heat
denatured at 70°C for 10 min in 0.2 M -mercaptoethanol, 0.5% SDS
and protease-inhibitor cocktail. Some samples were then incubated overnight at 37°C in an incubation solution (40 mM
Na2HPO4, pH = 7.4, 10 mM EDTA, 1%
-mercaptoethanol, and 0.6% Triton X-100) containing PNGase F (8,000 mU/ml) or endoglycosidase (Endo) F2 (3.2 mU/ml). Other
samples were treated overnight at 37°C in 200 mM sodium acetate
buffer (pH = 5) containing either Endo H (40 mU/ml) or
O-glycosidase (8 mU/ml). For membrane fractions extracted from oocytes,
pellets were dissolved in 20 µl of Na2HPO4
(40 mM, pH 7.0), and SDS was added to a final concentration of 1%.
After the membrane fractions were denatured at 70°C for 10 min, they were incubated at 37°C for 6 h in the presence of 5 U of
N-glycosidase F in the incubation solution. The reaction was stopped by
adding the SDS gel-loading buffer.
Lectin binding.
We used the following lectins: concanavalin A (Con A), wheat germ
agglutinin (WGA), glycine max agglutinin (soy bean agglutinin; SBA),
jacalin, Galanthus nivalis (GNA), Lens culinaris
(LCA), Ulex europaeus (UEA-I), and Tetragonobulys
purpurea (LTA). Lectins coupled to agarose beads (Sigma, St.
Louis, MO) were equilibrated at room temperature with the
lectin-binding buffer, which contained (in mM) 150 NaCl, 20 Tris, pH
7.5, 1 MgCl2, 1 MnCl2, 1 CaCl2, and
1 ZnSO4, 0.5% Triton X-100, and protease-inhibitor
cocktail. Agarose beads were then preabsorbed with 1% BSA in the
lectin-binding buffer for 5 min at RT, followed by three 10-min washes
in the lectin-binding buffer. Crude membrane proteins (50 µg) were
then added to the lectin-binding buffer containing lectin-agarose beads to a final volume of 200 µl. For competition experiments, sugars (methyl--D-mannopyranoside for Con A and GNA; galactose
for SBA and jacalin; N-acetyl-D-glucosamine for
WGA; and
-mannose for LCA) were added to a final concentration of
0.28 M. For UEA-1 and LTA,
-L-fucose was used to a final
concentration of 1 M. The reaction mixture, with or without competing
sugar, was incubated overnight at 4°C in a rotating shaker.
Afterward, the beads were washed three times with the lectin-binding
buffer for 10 min.
Immunoblotting. Membrane extracts treated with glycosidases or material from lectin-binding assays were mixed with Laemmli loading buffer and heated for 10 min at 70°C. The proteins were then separated on a 7.5% SDS-polyacrylamide gel and transferred overnight to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). Blots were preincubated for 1 h in a blocking buffer containing 5% nonfat dry milk and 0.1% Tween 20 in Tris-saline (TBS; 50 mM Tris, pH 7.4; 150 mM NaCl) and then incubated with an antibody (1:500 dilution), either NBC-5 (38) or K1A (6), which recognizes the COOH terminus of NBCe1-A. After several washes with TBS containing 0.1% Tween 20, blots were incubated with anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Chemicon) for 1 h (1:5,000 dilution). Blots were washed three times and developed by an HRP/hydrogen peroxide-catalyzed oxidation of luminol under alkaline conditions (Pierce, Rockford, IL).
Site-directed mutagenesis. The plasmid pTLN-NBCe1-A (35) served as a template for site-specific mutagenesis as described by Pusch et al. (33). Seven mutagenic primers were designed to replace the codons for Asn (N) at each of the putative glycosylation sites (N33, N199, N208, N497, N592, N597, and N617). We created a double (or triple) mutant with the same approach, but using a single (or double) mutant as the template. The final constructs were sequenced to verify the desired sequences.
Measurement of Vm and pHi.
Wild-type and mutant cDNA were transcribed in vitro using a mMessage
mMachine kit (Ambion, Austin, TX) with SP6 RNA polymerase. Defolliculated Xenopus oocytes (Stage V-VI) were prepared as
described previously and injected with 20 ng of cRNA or 50 nl water and incubated in OR3 media (50% Leibovitz L-15 media with
L-glutamine, 5 mM HEPES, pH 7.5) supplemented with 5 U/ml
penicillin/streptomycin (35). Injected oocytes were
maintained for 3-7 days at 18°C before use. Voltage and pH
microelectrodes were prepared as described previously
(17). The pH electrode tip was filled with proton ionophore 1 cocktail B (Fluka Chemical, Ronkonkoma, NY) and back filled
with a pH 7 phosphate buffer. Electrodes were connected to
high-impedance electrometers (model FD-223; WPI, Sarasota, FL), which
in turn were connected to the analog-to-digital converter of a
computer. In electrophysiological experiments, the
CO2/HCO
Statistics. Data are reported as means ± SE. Levels of significance were assessed using the unpaired, two-tailed Student's t-test. A P value of <0.05 was considered significant. Rates of pHi change (dpHi/dt) were fitted by a line using a least-square method.
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RESULTS |
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N-glycosylation of native NBCe1 in different species.
To test whether the N-glycosylation of NBCe1 is a general phenomenon
among various species, we first prepared membrane extracts prepared
from the kidneys of rabbit, rat, bovine, and salamander and then
treated these extracts with PNGase F. This enzyme digests the amide
bond between the reducing end of N-acetylglucosamine and the
-amide group of asparagine. We examined the effect of PNGase F
treatment on the MW of the NBCe1 protein by immunoblot analysis, using
an antibody specific to the COOH terminus of the protein. Figure
1 shows that PNGase F treatment reduced
the apparent MW of NBCe1 in all species examined. The immunoreactive
bands in rabbit, rat, and bovine decreased from ~130 to ~116, which is the size expected from the deduced amino acid sequence. Not shown
are data demonstrating that PNGase F treatment also reduces the
apparent MW of NBCe1 from ~130 to ~116 in mouse, human, and guinea
pig kidneys. These results suggest that N-glycosylation of NBCe1 is a
general phenomenon in mammals. As noted previously, salamander NBCe1
has an apparent MW of ~160 (38), which is 30 higher than
in mammalian species. Because PNGase F treatment reduced the apparent
MW of salamander NBCe1 to ~130, rather than the predicted MW of 116, it is possible that salamander kidney NBCe1 has an additional
O-glycosylation site(s).
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Glycosidase treatment of native rat NBCe1.
To investigate carbohydrate components of the protein, we focused on
rat kidney NBCe1, which has been characterized in terms of both its
functional properties (18, 34, 39) and its cellular localization (6, 28, 37, 38). In Fig.
2, lane 1 (no enzyme treatment) and lane 2 (PNGase F) confirm the rat data of
Fig. 1, which indicate that kidney NBCe1 has an N-glycosylated
oligosaccharide. To verify directly the absence of an O-glycosylated
carbohydrate-peptide linkage, we next treated rat kidney membrane
extracts with an O-glycosidase, which digests the bond between the
reducing end of N-acetyl-galactosamine (GalNAc) and the
hydroxyl group of serine or threonine. We found that treatment with a
combination of O-glycosidase and sialidase (separate experiments showed
that sialidase had no effect by itself) did not change the MW of the
protein, indicating absence of O-glycosylation (Fig. 2, compare
lanes 2 and 3). To further characterize the
carbohydrate component of NBCe1, we treated the protein with Endo
F2, which cleaves biantennary complex-type oligosaccharides, or Endo H, which cleaves oligosaccharides of the high
mannose and hybrid types. Because neither treatment shifted the MW of
NBCe1 (Fig. 2, lanes 4 and 5), we can conclude
that NBCe1 has an N-linked oligosaccharide that is probably of the tri-
or tetra-antennary type.
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Lectin binding of native rat NBCe1. Because a lectin affinity profile can provide some insight into the nature of the linked oligosaccharide, we performed lectin affinity chromatography with a series of lectins immobilized to agarose beads. We incubated crude membrane extracts with lectin-agarose beads, allowed glycoprotein to bind to the lectin, added either nothing or a competing monosaccharide (see MATERIALS AND METHODS) to elute bound glycoprotein from the beads, isolated the beads, eluted bound glycoprotein (if present) from the beads, and then performed an immunoblot on the supernatant.
As shown in Fig. 3, the NBCe1 protein bound to Con A, glycine max agglutinin (i.e., SBA), and WGA. Con A has an affinity for D-glucopyranose or D-mannopyranose with unmodified hydroxyl groups at the C3, C4, and C6 positions. Elution with methyl-
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Mutational analysis of consensus N-glycosylation sites.
As noted before, the amino acid sequence of rat kidney NBCe1-A predicts
seven consensus N-glycosylation sites (N-X-S/T): N33, N199, N208, N497,
N592, N597, and N617 (Fig. 4). To test
whether these consensus sites are responsible for N-glycosylation, we individually mutated each asparagine (N) to glutamine (Q). We then
heterologously expressed each mutant transporter in Xenopus oocytes, which are known to functionally express wild-type rat kidney
NBCe1-A (34). After extracting crude oocyte plasma
membranes, and treating some of them with PNGase F, we analyzed the
mutant proteins by immunoblotting. As shown in Fig.
5, wild-type NBCe1-A expressed in oocytes
and then either not treated or treated with PNGase F, had the same
apparent MWs (i.e., ~130 and ~116, respectively) as native NBCe1 in
mammalian tissues (see Figs. 1 and 2). The same is true for each of the
first five N-to-Q mutants. It is interesting to note that N592
(predicted to be on the third extracellular loop, 28 amino acids
downstream from the end of TM5) does not appear to be glycosylated
significantly in the wild-type NBCe1 protein.
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Functional expression of unglycosylated NBCe1.
To test the functional significance of the glycosylation of rat kidney
NBCe1, we expressed the triple mutant N592Q/N597Q/N617Q in
Xenopus oocytes and then used microelectrodes to monitor
pHi and membrane potential (Vm).
Previous work showed that, as expressed in Xenopus oocytes,
wild-type NBCe1-A is electrogenic, Na+ dependent, and
HCO
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DISCUSSION |
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Carbohydrate structure of rat kidney NBCe1.
In this study, we provide the first direct evidence for the
glycosylation of any of the Na+-coupled
HCO-linked galactose (insensitivity to jacalin); a terminal nonreducing
-linked mannose (insensitivity to GNA); or either a core or outer
fucose (insensitivity to LCA, LTA, and UEA-I). On the other hand, the carbohydrate component may include a terminal GalNAc (sensitivity to
SBA), either an internal GlcNAc or a terminal sialic acid (WGA sensitivity), and a D-glucopyranose or
D-mannopyranose with unmodified C3, C4, and C6 hydroxyl
groups (Con A sensitivity).
Glycosylation sites. Our data indicate that the N-linked glycosylation in NBCe1 normally occurs at N597 and N617 and thus prove that both of these residues, which are located between putative TM5 and TM6, face the extracellular fluid.
Glycosylation at N597 and N617 probably contributes about equally to the total glycosylation of the wild-type protein because 1) disrupting either N597 or N617 reduces the apparent MW of the mutant protein from ~130 to ~122, about one-half of the way to the MW of the unglycosylated protein; and 2) treatment of either mutant with PNGase F reduces the MW to ~116, the other one-half of the way. The N597Q/N617Q double mutant exhibits a pair of bands (MW of 116 and 120 in Fig. 6) that presumably represent the protein with or without glycosylation of N592. However, in the absence of PNGase F treatment, the N617Q single mutant exhibited only a single band (MW of 122), suggesting that glycosylation at N597 prevents glycosylation at N592. By steric hindrance, the bulky carbohydrate moiety at N597 may contribute to the absence of glycosylation at N592 in wild-type NBCe1. However, steric hindrance is not the entire explanation because, as noted above, glycosylation at N592 is optional even for single and double mutants in which we disrupt glycosylation at N597. One possibility is that N592, which is a 28-amino acid residue from the putative end of TM5, is too close to the membrane for efficient glycosylation. However, the N-glycosylation site on the fourth extracellular loop of AE1 is only 15 residues from the putative end of TM5. Alternatively, folding of the extracellular loop near N592 may make this asparagine an intrinsically poor substrate for the oligosaccharyl transferase in the endoplasmic reticulum.Retention of function in unglycosylated NBCe1.
The Xenopus oocyte expression system has been very useful
for analyzing the effect of glycosylation on the function of membrane proteins (25). In most of the membrane proteins studied to
date, such as the glutamate transporter GLT1 (40), the
-aminobutyric acid transporter GAT1 (26), and the
nicotinic acetylcholine receptor AChR (15), the naturally
occurring N-glycosylation is essential for functional expression of the
glycoprotein. Nonetheless, in other cases, such as the
Na+-dicarboxylate cotransporter NaDC-1 (29),
the Na-Pi cotransporter NaPi-2 (20), the water channel
aquaporin-1 (43), and the Na+ channel ENaC
(9), the N-glycosylation is not essential for protein
function. We found that mutations at one of the two natural sites
(N597Q or N617Q), at both of the two natural sites (N597Q/N617Q), or at
the two natural sites plus the one alternative site (N592Q/N597Q/N617Q) yield transporters, as expressed in Xenopus oocytes,
functionally comparable to the wild-type NBCe1 in terms of
electrogenicity as well as Na+ and HCO
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ACKNOWLEDGEMENTS |
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We thank D. Wong for computer support.
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FOOTNOTES |
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The work was supported by National Institutes of Health Grant DK-30344.
Present addresses: L. Hu, Leids Universitair Medisch Centrum, De Bleek 14, 2312 LP Leiden, The Netherlands; B. Schmitt, Anatomie I, Koellikerstr. 6, 97070 Wurzburg, Germany.
Address for reprint requests and other correspondence: I. Choi, Dept. of Physiology, Emory Univ. School of Medicine, 615 Whitehead Research Bldg., Atlanta, GA 30322 (E-mail: ichoi{at}physio.emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 25, 2003;10.1152/ajprenal.00131.2002
Received 9 April 2002; accepted in final form 14 February 2003.
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