Abstract
Abstract. This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with “studs,” which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.
Intercalated (IC) cells are prominent and characteristic cell types of the kidney collecting duct system (1,2,3,4,5). Two main types of IC cells have been distinguished: an acid-secreting A-type with a proton translocating ATPase in the apical cell membrane and a bicarbonate-secreting B-type with the proton translocating ATPase in the basolateral cell membrane (2, 5). The morphologic correlates of the cytoplasmic domain of the H+-ATPase in IC cells are so-called “studs,” hexagonal arrays, 10 nm in diameter, located at the cytoplasmic side of the cell membrane and of the membrane enclosing tubulovesicular profiles. These tubulovesicular profiles are quite characteristic for IC cells. They represent a cytoplasmic storage pool of proton pumps and are not clathrin-coated. If increased acid secretion is required, e.g., in the case of systemic acidosis, cytoplasmic H+-ATPase containing vesicles of A-type IC cells are shuttled to and inserted into the apical plasma membrane. A similar process appears to occur in B cells in the case of alkalosis, but is less well established at this site (6). Thus, modifications in proton secretion can be achieved without de novo synthesis of the enzyme. Although proton secretion occurs in other nephron segments as well, proton transport by a vacuolar-type H+-ATPase, which includes a shuttling mechanism from cytoplasmic stores to apical or basal cell membranes, is exclusively established in renal IC cells (3,4,5, 7, 8).
In this study, we report on a human autoantibody obtained from a woman with habitual abortion. In these patients, serum is routinely screened for irregular autoantibodies by immunohistochemical analysis of stomach, liver, and kidney. The serum of this patient showed a strong immunofluorescence staining of the kidney collecting duct system of rabbit, rat, and human (9). The patient showed no disorder of renal function as revealed by normal serum creatinine and blood urea nitrogen. We used this antiserum to characterize the corresponding antigen at cellular and molecular levels, which led to the identification of the GM2-activator protein (GM2AP) (10). In the brain, this protein is required for hexosaminidase A (Hex A) to hydrolyze the terminal β-GalNAc residue from ganglioside GM2 (10, 11). We discuss potential novel roles of GM2AP in the regulation of IC cell function.
Materials and Methods
Antibodies
Primary antibodies used in this study were a human autoantibody obtained from a woman with habitual abortion, a polyclonal rabbit antibody raised against a glutathione S-transferase-GM2 activator fusion protein as described previously (12), and the monoclonal antibody E11 directed against the 31-kD subunit of the H+-ATPase (13, 14).
Tissues for Immunohistochemistry
Adult female Sprague Dawley rats (200 g body wt) were perfused via the abdominal aorta with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 3 min at 220 mmHg followed by cryoprotectant sucrose-PBS solution (800 mosmol) for 5 min at 220 mmHg. Organs were removed, and slices of tissues were frozen in isopentane cooled by liquid nitrogen to -160°C.
Immunofluorescence Microscopy
Immunofluorescence staining was performed on 4-μm frozen sections and on semithin frozen sections from the following rat organs: adrenal gland, aorta, brain, colon, esophagus, heart, jejunum, kidney, liver, lung, spleen, ovary, placenta, skeletal muscle, stomach, thyroid gland, and uterus. After rinsing with PBS, unspecific binding sites were blocked with 2% fetal calf serum (FCS), 2% bovine serum albumin (BSA), and 0.2% fish gelatin in PBS for at least 30 min. Primary antibodies (prediluted in blocking solution) were applied for 60 min at room temperature. Antigen-antibody complexes were visualized using fluorochrome (Cy2 or Cy3)-conjugated secondary antibodies (Biotrend, Cologne, Germany; prediluted in blocking solution). Sections were washed with PBS, rinsed with H2O, and mounted in 15% mowiol (Calbiochem, Bad Soden, Germany) and 50% glycerol in PBS. After overnight drying, specimens were analyzed and documented with a Polyvar 2 photomicroscope (Leica, Bensheim, Germany).
Simultaneous confocal double fluorescence microscopy was done with a Zeiss 410 ultraviolet laser scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with appropriate filters. Micrographs were taken using an image recorder (Focus Graphics, Foster City, CA) and digitally processed using the Adobe Photoshop 4.0 program.
Immunoelectron Microscopy
For preembedding immunoperoxidase-labeling, cryostat sections of perfusion-fixed tissues from rat kidney cortex were cut at 20 μm and processed as free-floating sections. After preincubation in PBS containing 10% normal swine serum, the human autoantibody (prediluted 1:16 in PBS containing 10% normal swine serum) was applied overnight at room temperature followed by biotinylated goat antihuman IgG (1:50) for 1 h. After washing with PBS, sections were placed in streptavidin-conjugated peroxidase complexes (1:200). Peroxidase activity was detected using 3,3′-diaminobenzidine as chromogen. The sections were stained en bloc with uranyl acetate in maleate buffer, osmicated in 1% OsO4 for 1 h, dehydrated in graded series of acetone, and flat-embedded in Epon between Teflon-coated slide and coverglass. Areas of interest were selected under a light microscope, excised with a razor blade, and mounted onto preformed Epon blocks. Thin sections were cut with an ultramicrotome E (Leica) and examined with a Philips EM 301 microscope (Munich, Germany).
Immunogold labeling of ultrathin frozen sections from rat kidney cortex was performed as described previously (15). During the incubation, the grids were kept floating on drops of filtered solutions in a moist chamber. After washing with PBS, unspecific binding sites were blocked with 10% FCS in PBS containing 10 mM glycine for 1 h at room temperature. The primary antibody was applied in an adequate dilution overnight at +4°C. After rinsing with washing buffer (PBS containing 0.1% BSA), rabbit-antihuman-IgG (Zymed, Aidenbach, Germany) was applied at 1:50 (diluted with 10% FCS in PBS) for 1 h at room temperature as a bridge, followed by a goat-anti-rabbit-IgG coupled to 10 nm colloidal gold (Biocell; Plano, Marburg, Germany) at 1:100 in PBS containing 0.1% acetylated BSA. After rinsing with washing buffer (10 times for 3 min) and PBS (twice for 3 min), the sections were post-fixed with 2% glutaraldehyde for 10 min and stained with 1% tannic acid followed by 2% OsO4. After staining with 2% uranyl acetate for 2 to 5 min, the sections were finally adsorption-stained with 0.003% lead citrate in 2% polyvinyl alcohol (molecular weight 10,000; Sigma) (16). After air drying, the sections were analyzed under a Philips EM 301 microscope.
Protein Extraction for Immunoblotting and Protein Purification
Kidneys from adult female Sprague Dawley rats (200 g body wt) were harvested, and kidney cortex was immediately frozen in liquid nitrogen. Samples were stored at -80°C until use. Protein extraction was carried out at +4°C in a tight-fitting Potter homogenizer with 15 strokes at 1400 rpm in 10 vol of homogenization buffer (20 mM Tris, 500 mM NaCl, pH 7.5) supplemented with 0.5% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS; Sigma). Insoluble material was pelleted at 40,000 × g for 30 min at +4°C. The resulting supernatant was stored at -20°C.
Gel Electrophoresis, Immunoblotting, and Protein Sequence Analysis
Protein extracts were diluted with sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, and separated on 10% SDS polyacrylamide gels. Proteins were transferred to Immobilon P membranes (Millipore, Eschborn, Germany) by semidry blotting and stained with 0.05% Coomassie Brilliant Blue in 40% methanol and 7% acetic acid. Before immunodetection, membranes were destained with methanol. After blocking unspecific binding sites with blocking solution (5% milk in PBS) for 30 min at room temperature, primary antibodies (either human autoantibody or rabbit polyclonal anti-GM2AP, diluted in blocking solution) were used at 1:500 dilution, and horseradish peroxidase-conjugated secondary antibodies (Promega, Heidelberg, Germany) were used at 1:20,000 dilution. The immunoreaction was visualized by chemiluminescence and film exposure.
For protein sequencing, the antigen was enriched by preparative HPLC reversed-phase chromatography, and purification was monitored by Western blotting. Per run 1 ml of the supernatant (see above) was loaded onto a Brownlee 4.6-mm C-8 column (Applied Biosystems, Weiterstadt, Germany) and developed with a linear gradient of 10 to 80% acetonitrile during 45 min. Two-milliliter fractions were collected, and 0.1-ml aliquots were lyophylized, resuspended in SDS sample buffer, and analyzed by immunoblotting. Positive fractions were lyophylized, resuspended in 100 μl of lysis buffer, and separated by two-dimensional gel electrophoresis with NEPHGE (nonequilibrium pH gradient gel electrophoresis) in the first dimension, followed by separation in 10% SDS gels in the second dimension as described (17). After transfer to Immobilon P membranes, protein spots were excised from Coomassie Brilliant Blue-stained membranes. Tryptic digestion of excised spots, HPLC separations of resulting internal peptide fragments, and peptide microsequencing were done as described (17), except that Edman degradation was carried out on a Procise 494 protein sequencing apparatus (Applied Biosystems).
Control Experiments
The controls for all immunodetection steps were done by replacing the primary antibodies with either normal human serum or blocking solution. In all cases, no unspecific background staining was observed.
Results
Molecular Identification of the Autoantibody-Defined Antigen as Ganglioside GM2-Activator Protein
A human serum obtained from a woman with habitual abortion containing an undefined antibody, previously shown to react with renal IC cells (9), was used to characterize the corresponding antigen at the cellular and molecular level. In Western blot experiments of cytosolic extracts from rat kidney cortex, a protein with an apparent molecular weight of 23 kD was detected (Figure 1, lane 1). This protein was enriched by one step of HPLC reversed-phase chromatography. Positive fractions were separated by preparative two-dimensional gel electrophoresis. Protein spots were excised from Coomassie Brilliant Blue-stained preparative two-dimensional blots and subjected to tryptic digestion (Figure 2, a and b). Two of the resulting internal peptides of the rat protein were analyzed by Edman degradation. Database comparison revealed that these peptides matched for 100% with corresponding sequences of ganglioside GM2-activator protein (Figure 3), thus identifying the antigen recognized by the human serum as GM2AP.
Western blot analysis of 0.5% CHAPS extract from rat kidney cortex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10% polyacrylamide gels. The immunoreaction was detected by a horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence system. The human autoantibody (lane 1) and the rabbit polyclonal anti-GM2AP-antibody (lane 2) detect the same 23-kD protein band. Lanes 1′ and 2′ show the staining of the identical membrane (from lanes 1 and 2) with Coomassie Brilliant Blue to demonstrate protein loading and separation. The protein band at approximately 40 kD (lane 2) appears to be nonspecific probably due to large amounts of loaded protein.
Two-dimensional analysis of rat kidney GM2AP. (a) Coomassie Brilliant Blue-stained two-dimensional Western blot using nonequilibrium pH gradient gel electrophoresis (NEPHGE) in the first dimension, and SDS-PAGE in the second, of a fraction enriched for GM2AP (arrow) obtained after reversed-phase HPLC of a supernatant from rat kidney cortex cytosolic extracts. (b) Enhanced chemiluminescence detection of GM2AP using the human autoantibody. Arrows in a and b indicate the excised and sequenced protein spot.
Amino acid sequence of the mouse GM2 activator protein (AC U09816). Peptide sequences obtained from Edman degradation of excised protein spots from two-dimensional Western blots of rat kidney cortex extracts are underlined and show 100% identity with GM2AP.
In Western blot experiments with a rabbit polyclonal antiserum directed against GM2AP, the same 23-kD band was detected in extracts from rat kidney cortex as that recognized by the human autoantibody (Figure 1, lane 2).
Western blot experiments with the purified proton pump revealed no immunoreactivity with the human autoantibody, indicating that GM2AP is not a part of the proton pump complex (S. Gluck, Gainesville, FL, personal communication).
GM2AP Is Associated with the Studs of Tubulovesicular Profiles in Collecting Duct IC Cells
To determine the distribution of GM2AP recognized by the human autoantibody in the kidney, we performed immunofluorescence stainings of sagittal and coronal serial sections from adult rat kidney using the human serum as first antibody. The immunoreactivity was restricted to the collecting duct system (Figure 4b). All IC cells of the collecting duct were heavily labeled, whereas adjacent principal cells were not stained. No additional labeling of other nephron segments was observed. No immunoreactivity was found in other H+-ATPase-expressing organs (e.g., stomach and colon). On semithin frozen sections of rat kidney cortex, three distinct patterns of immunostaining within IC cells could be observed corresponding to the localization pattern of the proton pumping ATPase in IC cells: staining of the apical cell pole, staining of the basolateral plasma membrane, and a diffuse cytoplasmic labeling (Figure 4b). To demonstrate the association of GM2AP with the proton pump, we performed double-labeling experiments using a monoclonal antibody against the proton pump (Figure 4a) and the human autoantibody (Figure 4b) showing a complete overlap of immunoreactivity. To reveal the subcellular localization of GM2AP within IC cells, we performed preembedding immunoperoxidase labeling experiments of rat kidney cortex. The electron-dense reaction product was restricted to IC cells (Figure 5a). At higher magnification, the immunostaining was found at the cytoplasmic aspect of the apical cell membrane and of the enclosing membrane of tubulovesicular profiles (Figure 5b). High resolution electron microscopy revealed the association of GM2AP with the studs of the apical cell membrane and of tubulovesicular profiles of IC cells (Figure 5c). To a much weaker extent, the immunoreaction was also found at the basolateral cell membrane (data not shown). The cytosolic localization of GM2AP within the IC cells was also seen after immunogold labeling of ultrathin frozen sections of rat kidney cortex (Figure 6). The gold particles were found at the cytoplasmic aspect of the apical cell membrane and of the enclosing membrane of tubulovesicular profiles. The gold particles (arrows) are clearly located at the outer leaflet of the bilayer. No immunoreactivity was found in the lumen of tubulovesicular profiles.
Double fluorescence analysis of rat kidney cortex. Semithin (0.5 μm) frozen section with the monoclonal anti- H+-ATPase antibody (a) and the human autoantibody (b) reveals 100% overlap of immunoreactivity indicating coexpression of GM2AP and the vacuolar type H+ ATPase in intercalated (IC) cells. Staining of the apical plasma membrane (arrowhead), basolateral plasma membrane (arrow), and diffuse cytoplasmic labeling corresponding to the localization pattern of the proton pumping ATPase in IC can be observed. Magnification: ×100 in a and b.
Immunoelectron microscopic analysis of GM2AP. The subcellular localization of the antigen recognized by the human autoantibody in IC cells was analyzed by preembedding immunoperoxidase labeling. (a) At low magnification, strong immunostaining of the mitochondria-rich IC cell (IC) is obvious, whereas the adjacent principal cell of collecting duct epithelium (PC) is not labeled. (b) At higher magnification, the immunoreaction can be localized to the inner aspect of the apical cell membrane (arrows) and to cytoplasmic tubulovesicular profiles (arrow-heads). (c) At high resolution, GM2AP is shown to be associated with the cytoplasmic studs of tubulovesicular profiles. (d) After replacing the primary antibody with blocking solution, no labeling is observed. Magnification: ×7,100 in a; ×15,000 in b; ×45,000 in c; ×9,100 in d.
Immunogold labeling of an ultrathin frozen section of rat kidney cortex showing the cytosolic localization of GM2AP within the IC cells. The gold particles are found at the cytoplasmic aspect of the apical cell membrane and of the enclosing membrane of tubulovesicular profiles. The gold particles (arrows) are clearly located at the outer leaflet of the bilayer. Note the absence of labeling from the lumen of tubulovesicular profiles. Adjacent mitochondria were not labeled, indicating the specificity of the immunoreaction. Magnification, ×49,000.
To further confirm the identity of the autoantibody-defined antigen as GM2AP, we performed confocal laser scanning double fluorescence microscopy of rat kidney cortex sections. The human serum (Figure 7a) and a polyclonal rabbit antibody raised against GM2AP (Figure 7b) revealed a complete overlap of immunoreactivity (Figure 7c).
Confocal laser scanning double fluorescence analysis of rat kidney cortex. The incubation of the same frozen section with the human autoantibody (a) and a polyclonal rabbit anti-GM2-activator antibody (b) reveals 100% overlap of immunoreactivity. (c) After superimposing both channels, a yellow color results from the overlap of both signals. This 100% overlap indicates that the labeling pattern of both antibodies is identical, proving that the antigen recognized by the human antibody is ganglioside GM2 activator protein. Magnification: ×63 in a through C.
Discussion
A human autoantibody obtained from a woman with habitual abortion was previously found to react with IC cells of the kidney collecting duct system (9). In the present study, we confirmed and extended these findings with respect to the various types of IC cells, the subcellular distribution of the recognized antigen, and the molecular identity of the antigen.
Several distinct patterns of immunoflourescence staining in IC cells were observed: exclusive staining of the apical cell pole, diffuse staining of the entire cytoplasm, and basolateral dominance. This labeling pattern is identical to that of the vacuolar type H+-ATPase in the various types of IC cells, i.e., acid-secreting A-type, bicarbonate-secreting B-type, and an intermediate type of IC cells (14, 18). The immunoreactivity of the human autoantibody was restricted to IC cells. No other nephron segment and no other proton pump-expressing tissues examined were reactive.
On the subcellular level, we could demonstrate by preembedding immunoperoxidase and immunogold labeling that within the IC cells the antigen was associated with so-called studs of tubulovesicular profiles, and the cell membrane at the apical and in a much weaker extent at the basolateral localization. The weaker extent of basolateral labeling is in line with the previous description that studs are very rarely found at the basolateral domain (1). Previous studies had shown that the vesicle-coating studs of IC cells do not contain clathrin but represent the cytoplasmic subunits of the proton pump containing the 31-, 56-, and 70-kD subunits of this enzyme (19,20,21,22). This proton pump can be shuttled between the plasma membrane and tubulovesicular profiles that serve as a storage pool of proton pumps in IC cells. Although the enzyme is also found in other segments of the nephron, only collecting duct IC cells are equipped with a transportable type of proton pump. Western blots from cytosolic extracts from rat kidney cortex with the human autoantibody revealed a 23-kD protein. Western blot analysis with the purified proton pump showed no immunoreactivity with the human autoantibody, indicating that this 23-kD protein does not belong to the proton pump complex itself but, most likely, is associated with domains involved in the vesicular shuttling of the pumps. In addition, it became clear from these experiments that GM2AP is not tightly associated with the proton pump.
Protein purification and amino acid sequence analysis of internal peptide fragments generated by tryptic digestion identified the 23-kD protein as the ganglioside GM2-activator protein (GM2AP). These findings were confirmed by immunohistochemical and biochemical experiments. A polyclonal rabbit antibody raised against GM2AP showed the same reaction pattern in immunohistochemical and in Western Blot experiments as the human autoantibody.
Sphingolipids are important components of the outer leaflet of eukaryotic cell membranes (10, 11). Gangliosides are glycosphingolipids containing one or more sialic acid residues. They are degraded by sequential action of acid lysosomal glycosidases. Several inherited deficiencies of individual catabolic steps exist in humans and result in a lethal accumulation of a glycosphingolipid, leading to the clinical course of gangliosidosis. One such step is the hydrolyzation of terminal β-Gal-NAc-residues from ganglioside GM2 by human β-hexosaminidase A (Hex A). This reaction requires the correct synthesis and combination of three gene products. Two of these encode the alpha (HEXA) and beta (HEXB) subunits of Hex A, and the third encodes a small, nonenzymatic, substrate-specific cofactor, the GM2-activator protein (GM2AP), a sphingolipid binding protein. Mutations in any one of these genes can result in one of the three diseases collectively known as the GM2 gangliosidoses (HEXA, B variant or Tay-Sachs disease; HEXB, O variant or Sandhoff disease; and GM2A, AB variant) (reviewed in reference (23)). The amounts of GM2AP in human liver, spleen, and placenta range from 50 to 120 ng/mg, whereas the concentration of GM2AP found in the kidney is extremely high, with levels up to 800 ng/mg (10), although its established substrate ganglioside GM2 and its precursor GM1 are not significantly expressed in the kidney (24, 25).
A proposed mode of action of GM2AP is that the activator binds a single GM2 molecule and lifts it out of the membrane so that this activator-lipid complex can be reached and recognized by water-soluble Hex A (10). As GM2AP binds tightly to octyl-Sepharose, a hydrophobic binding site is assumed to be present in this protein (10). GM2AP was further shown to transfer glycolipids from one liposome to another in the absence of Hex A (11, 26).
So far GM2AP has never been considered in any kidney function. We show that GM2AP is associated with the proton pump of IC cells, most likely not with the pump itself but with the machinery involved in shuttling of the pump.
The shuttling mechanism of the proton pump in IC cells is not fully understood. However, an involvement of microtubules was demonstrated, as vesicular transport can be inhibited by approximately 70% after in vivo depolymerization with microtubule-disrupting agents such as colchicine (19, 21, 27). The involvement of other proteins, such as GTP-binding proteins or vesicle-associated membrane proteins, is not clear at all (28). It has been shown that some GTP-binding proteins are associated with the H+-ATPase and can regulate its function (7, 29), whereas some IC cells, which show a prominent expression of the proton pump at the apical membrane, do not stain for any G protein α-subunit. Another study proposed that some G proteins may play an important role not only in the regulation (transport/activity) of the proton pump, but also in targeting the pump to the cell membrane (30).
Our data suggest a novel intracellular location and role for GM2AP in the kidney as a part of the vesicular transport/fusion mechanisms in IC cells. We propose an involvement of GM2AP not in the transport of H+ itself, but possibly in shuttling of the enzyme.
Because of the presence of an N-terminal signal peptide, N-linked glycosylation, and disulfide bonds, it is clear that the activator is synthesized in the endoplasmic reticulum (reviewed in reference (31)). Thus, our finding of the activator on the cytosolic aspect of the vesicular membrane and on the cytoplasmic aspect of the cell membrane is surprising. However, in their study of endosomal-lysosomal fusion, Kuwana et al. mistakenly identified the activator as a mediator of membrane fusion (32). They later discovered that the activator protein could “leak” from their lysosomal preparation and act as a transporter of the hydrophobic dye R-18 (used to measure membrane fusion) between labeled and unlabeled vesicles (33). Our present data suggest that this in vitro leakage may represent an uncharacterized in vivo transport process allowing the activator to cross the lysosomal and/or endosomal membranes.
With regard to the subcellular distribution of GM2AP, an exciting observation was made. Previously, it had been generally assumed that lysosomal proteins have an anticytosolic localization to protect the cells from autodigestion. We can now clearly demonstrate by immunogold electron microscopy that GM2AP has a cytoplasmic localization in IC cells. These findings are in line with the idea that GM2AP may have a novel role in IC cells of the kidney, which might be different from its role as a cofactor of Hex A. Since we can clearly demonstrate a cytosolic localization of GM2AP in IC cells, we propose a novel cytoplasmic role for GM2AP within the IC cells.
There are several possible modes of action for GM2AP in IC cells: (1) GM2AP could act in its established function as activator of Hex A in the degradation pathway of GM2. However, because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney (24) and because GM2AP has a cytosolic localization in IC cells that is not compatible with its intravesicular localization in serving as a cofactor for HEX A, this option appears rather unlikely. (2) GM2AP could be involved in linking the proton pumps to microtubules. (3) GM2AP might also be involved in regulating the activity of the proton pumps in IC cells. (4) Although in general GM2AP serves to bind a single GM2 molecule and to lift it out of the membrane (10), in IC cells it may bind polarized lipids of the cell membrane, which have been recently shown to participate in the targeting of nonpolarized proteins (34). In line with the latter study, GM2AP could be one of the postulated mediators of vesicular traffic in IC cells (5). Recently, the generation of a GM2AP-deficient mouse strain has been reported, which shows cerebellar pathology and motor impairment (35). These mice should prove helpful in further studying the participation of GM2AP in acid-base metabolism of IC cells.
Acknowledgments
Acknowledgments
This work was supported by a grant (Juniorantrag 90/95) from the Medizinische Fakultät, Ruprecht-Karls-Universität Heidelberg, to Dr. Peter Mundel. We thank Alexandra Zeller and Hiltraud Hosser for expert technical assistance, as well as Ingrid Ertl and Rolf Nonnenmacher for skillful photographic work and artwork. We are thankful to Dr. S. Gluck (Gainesville, FL) for providing the monoclonal antibody E11 directed against the 31-kD subunit of the vacuolar type H+-ATPase.
Footnotes
American Society of Nephrology
This work has been presented in part at the 30th annual meeting of the American Society of Nephrology, San Antonio, Texas, November 2 to 5, 1997. Dr. Thomas M. Mundel's present address: Department of Surgery, University of Münster, Münster, Germany.
- © 1999 American Society of Nephrology
