A Non-sulfated Form of the HNK-1 Carbohydrate Is Expressed in Mouse Kidney
2005; Elsevier BV; Volume: 280; Issue: 25 Linguagem: Inglês
10.1074/jbc.m501728200
ISSN1083-351X
AutoresHideki Tagawa, Yasuhiko Kizuka, Tomoko Ikeda, Satsuki Itoh, Nana Kawasaki, Hidetake Kurihara, Maristela L. Onozato, Akihiro Tojo, Tatsuo Sakai, Toshisuke Kawasaki, Shogo Oka,
Tópico(s)Caveolin-1 and cellular processes
ResumoThe HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Galβ1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin α and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate. The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Galβ1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin α and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate. IntroductionGlycosylation is one of the major post-translational protein modifications that play important roles in a variety of cellular functions, including recognition and adhesion. We have been interested in the HNK-1 (human natural killer-1) carbohydrate, which is recognized by anti-HNK-1 monoclonal antibody (1Abo T. Balch C.M. J. Immunol. 1981; 127: 1024-1029PubMed Google Scholar). The carbohydrate is expressed predominantly in the nervous system in a wide range of species, and its expression is spatially and temporally regulated during development of the nervous system (2Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (141) Google Scholar, 3Yoshihara Y. Oka S. Watanabe Y. Mori K. J. Cell Biol. 1991; 115: 731-744Crossref PubMed Scopus (57) Google Scholar). The characteristic structural feature of the carbohydrate is a sulfated trisaccharide, HSO3-3GlcAβ1-3Galβ1-4GlcNAc 1The abbreviations used are: GlcA, glucuronic acid; GlcAT, glucuronyltransferase; mAb, monoclonal antibody; AQP, aquaporin; pAb, polyclonal antibody; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MSn, multistage tandem mass spectrometry. (4Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 5Voshol H. van Zuylen C.W. Orberger G. Vliegenthart J.F. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and the inner structure, Galβ1-4GlcNAc, is commonly found on various glycoproteins and glycolipids. Therefore, glucuronyltransferase(s) and sulfotransferase(s) are supposed to be key enzymes for the biosynthesis of this carbohydrate (6Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) that are involved in the biosynthesis of the HNK-1 carbohydrate from rat, mouse, and human (7Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 8Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar, 9Yamamoto S. Oka S. Saito-Ohara F. Inazawa J. Kawasaki T. J. Biochem. (Tokyo). 2002; 131: 337-347Crossref PubMed Scopus (21) Google Scholar, 10Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 65: 166-173Crossref PubMed Scopus (45) Google Scholar, 11Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To elucidate the function of the HNK-1 carbohydrate, we generated mice with a targeted deletion of the GlcAT-P gene (12Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The HNK-1 carbohydrate has disappeared almost completely in the nervous system of GlcAT-P gene-deficient mice, and these mice exhibit reduced long-term potentiation at the Schaffer collateral CA1 synapses and defects in spatial memory formation. However, a trace of anti-HNK-1 antibody immunoreactivity remains on the surfaces of the soma and proximal dendrites of a subset of neurons in some limited regions. The remaining HNK-1 carbohydrate in GlcAT-P gene-deficient mice is assumed to be synthesized by GlcAT-S. More recently, we characterized the acceptor specificities of the two glucuronyltransferases using various oligosaccharides (13Kakuda S. Sato Y. Tonoyama Y. Oka S. Kawasaki T. Glycobiology. 2005; 15: 203-210Crossref PubMed Scopus (26) Google Scholar). The results suggested the possibility that the two glucuronyltransferases synthesize structurally and functionally different HNK-1 carbohydrates.To elucidate the function of the HNK-1 carbohydrate synthesized by GlcAT-S, we examined GlcAT-S mRNA expression in various mouse tissues, including brain. To our surprise, Glc-AT-S mRNA was expressed at higher levels in the kidney than in the brain. In this study, we provide the first evidence that the HNK-1 carbohydrate is synthesized by GlcAT-S as the non-sulfated form in mouse kidney. The non-sulfated form of HNK-1 carbohydrate was expressed predominantly on meprin α and CD13/aminopeptidase N. We also examined in detail the distribution of the carbohydrate in mouse kidney. The results are important for understanding the functions of the HNK-1 carbohydrate other than those in the nervous system.DISCUSSIONThe HNK-1 carbohydrate is expressed at high levels in the nervous system. The HNK-1 carbohydrate structure on P0 and the neural cell adhesion molecule, the major carrier glycoproteins in the peripheral and central nervous systems, respectively, has been determined (27Gallego R.G. Blanco J.L. Thijssen-van Zuylen C.W. Gotfredsen C.H. Voshol H. Duus J.O. Schachner M. Vliegenthart J.F. J. Biol. Chem. 2001; 276: 30834-30844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Wuhrer M. Geyer H. von der Ohe M. Gerardy-Schahn R. Schachner M. Geyer R. Biochimie (Paris). 2003; 85: 207-218Crossref PubMed Scopus (36) Google Scholar, 29Liedtke S. Geyer H. Wuhrer M. Geyer R. Frank G. Gerardy-Schahn R. Zahringer U. Schachner M. Glycobiology. 2001; 11: 373-384Crossref PubMed Scopus (86) Google Scholar). All of the HNK-1 carbohydrate structure determined was of the sulfated form, i.e. sulfoglucuronic acid attached to N-acetyllactosamine, the non-sulfated form of the HNK-1 carbohydrate not being found on these molecules. These results indicate that almost all of the HNK-1 carbohydrate in the nervous system is expressed as the sulfated form. This is the first report that the HNK-1 carbohydrate is expressed at high levels in mouse kidney as the non-sulfated form. We have also demonstrated that GlcAT-S (but not GlcAT-P) is involved mainly in the expression of the non-sulfated HNK-1 carbohydrate in mouse kidney.Two glucuronyltransferases (GlcAT-P and GlcAT-S) have so far been identified as HNK-1 biosynthetic enzymes (7Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 8Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar, 9Yamamoto S. Oka S. Saito-Ohara F. Inazawa J. Kawasaki T. J. Biochem. (Tokyo). 2002; 131: 337-347Crossref PubMed Scopus (21) Google Scholar, 10Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 65: 166-173Crossref PubMed Scopus (45) Google Scholar, 11Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We have generated GlcAT-P gene-deficient mice and demonstrated that almost all of the HNK-1 carbohydrate in the brain is synthesized by GlcAT-P because of the absence of the HNK-1 carbohydrate in GlcAT-P gene-deficient mouse brain (12Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We have also demonstrated that the HNK-1 carbohydrate synthesized by GlcAT-P is involved in synaptic plasticity and in spatial learning (12Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, little is known about the function of the HNK-1 carbohydrate regulated by GlcAT-S. It should be noted that these two enzymes have significantly different acceptor specificities. Thus, GlcAT-P specifically recognizes N-acetyllactosamine (Galβ1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognizes not only the terminal Galβ1-4GlcNAc structure, but also the Galβ1-3GlcNAc structure, and shows the highest activity to-ward triantennary N-linked oligosaccharides (13Kakuda S. Sato Y. Tonoyama Y. Oka S. Kawasaki T. Glycobiology. 2005; 15: 203-210Crossref PubMed Scopus (26) Google Scholar), suggesting that they may synthesize structurally and functionally different HNK-1 carbohydrates. To elucidate the function of the HNK-1 carbohydrate synthesized by GlcAT-S, it is important that GlcAT-S is expressed not only in the brain, but also in the kidney.Immunofluorescence staining of mouse kidney with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex, where the GlcAT-S mRNA was also detected (Figs. 7 and 10). The non-sulfated HNK-1 carbohydrate was expressed at high levels in the brush borders of the S2 and S3 segments of the proximal tubules. In comparison, no significant signal for antibody M6749 was observed in the S1 segment immediately after the glomerulus (Fig. 8). Protein kinases Cα and Cβ1 are another case of such segment-specific expression. The two isoforms of protein kinase C are localized only to the brush borders of the S2 and S3 segments of proximal tubules, whereas the S1 segment is negative (30Pfaff I.L. Wagner H.J. Vallon V. J. Am. Soc. Nephrol. 1999; 10: 1861-1873PubMed Google Scholar). It is difficult to distinguish the segments of the proximal tubules with respect to protein absorption under normal conditions. However, lysosomal enzyme activities are increased in the S2 and S3 segments of proteinuric animals because of an increase in protein uptake (31Olbricht C.J. Cannon J.K. Garg L.C. Tisher C.C. Am. J. Physiol. 1986; 250: F1055-F1062PubMed Google Scholar). Therefore, the S2 and S3 segments might be more important under pathological conditions. In addition, antibody M6749 immunoreactivity was also detected in the thin ascending limb in the inner medulla, where no detectable level of either GlcAT-S or GlcAT-P mRNA was observed. These results suggest that the non-sulfated HNK-1 carbohydrate detected in the inner medulla could be synthesized by another glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate. The carbohydrate structure expressed in the thin ascending limb could also be different from those on CD13/aminopeptidase N and meprin α expressed in proximal tubules.The meprin α-subunit and CD13/aminopeptidase N were identified as major carrier proteins of the non-sulfated HNK-1 carbohydrate. Meprins (α and β) are members of the astacin family of metalloproteases (32Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (349) Google Scholar). Meprins are capable of activation or degradation of bioactive peptides, growth factors, cytokines, hormones, and matrix proteins through limited proteolysis (33Villa J.P. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2003; 278: 42545-42550Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Meprin α contains 10 potential N-linked glycosylation sites and is extensively glycosylated; ~25% of the total molecular mass is carbohydrate (34Tang J. Bond J.S. Arch. Biochem. Biophys. 1998; 349: 192-200Crossref PubMed Scopus (31) Google Scholar). CD13/aminopeptidase N is also an abundant metalloprotease in the brush borders of kidney proximal tubule cells (35Tauc M. Chatelet F. Verroust P. Vandewalle A. Poujeol P. Ronco P. J. Histochem. Cytochem. 1988; 36: 523-532Crossref PubMed Scopus (38) Google Scholar) and is the enzyme responsible for the extracellular conversion of angiotensin III to angiotensin IV (36Zini S. Fournie-Zaluski M.C. Chauvel E. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11968-11973Crossref PubMed Scopus (286) Google Scholar). CD13/aminopeptidase N also has 10 potential N-glycosylation sites. Thus, the non-sulfated HNK-1 carbohydrate is commonly expressed on two major metalloproteases that are extensively glycosylated in the kidney. It has been reported that N-linked carbohydrates on meprin α are required for optimal enzyme activity and for secretion, as found in a series of mutation analyses of N-glycosylation sites (37Kadowaki T. Tsukuba T. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2000; 275: 25577-25584Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). These results suggest that the non-sulfated HNK-1 carbohydrate may be involved in the regulation of metalloprotease activity, which leads to the functional diversity of these two molecules.Meprin α and CD13/aminopeptidase N are expressed on the apical membranes of the proximal tubules in the kidney cortex, but not in the inner medulla (33Villa J.P. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2003; 278: 42545-42550Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 35Tauc M. Chatelet F. Verroust P. Vandewalle A. Poujeol P. Ronco P. J. Histochem. Cytochem. 1988; 36: 523-532Crossref PubMed Scopus (38) Google Scholar). We also confirmed that anti-CD13/aminopeptidase N antibody immunoreactivity is not detected in the inner medulla (data not shown). Therefore, the immunoreactivity of antibody M6749 in the inner medulla is not due to the non-sulfated HNK-1 carbohydrate expressed on meprin α and CD13/aminopeptidase N, but might be due to that expressed on minor glycoproteins (~90 and 180 kDa), which were detected upon Western blot analysis, as shown in Fig. 5A. Another possibility is that the non-sulfated HNK-1 carbohydrate is expressed on glycolipids.The characteristic structural feature of the HNK-1 carbohydrate is sulfated glucuronic acid (4Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 5Voshol H. van Zuylen C.W. Orberger G. Vliegenthart J.F. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The important functional roles of the sulfate residue have been reported. Thus, the HNK-1 carbohydrate binds to laminin, but this binding is completely abolished upon desulfation of this carbohydrate (38Mohan P.S. Chou D.K. Jungalwala F.B. J. Neurochem. 1990; 54: 2024-2031Crossref PubMed Scopus (60) Google Scholar, 39Schmitz B. Schachner M. Ito Y. Nakano T. Ogawa T. Glyco-conj. J. 1994; 11: 345-352Crossref PubMed Scopus (43) Google Scholar). HNK-1 sulfotransferase gene-deficient mice exhibit similar phenotypes compared with GlcAT-P gene-deficient mice, including a reduction in long-term potentiation and a defect in spatial learning (12Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 40Senn C. Kutsche M. Saghatelyan A. Bosl M.R. Lohler J. Bartsch U. Morellini F. Schachner M. Mol. Cell. Neurosci. 2002; 20: 712-729Crossref PubMed Scopus (72) Google Scholar). Recently, interesting evidence has been reported concerning the functional role of glucuronic acid in the HNK-1 carbohydrate. Senn et al. (40Senn C. Kutsche M. Saghatelyan A. Bosl M.R. Lohler J. Bartsch U. Morellini F. Schachner M. Mol. Cell. Neurosci. 2002; 20: 712-729Crossref PubMed Scopus (72) Google Scholar) confirmed that the non-sulfated HNK-1 carbohydrate is expressed in HNK-1 sulfotransferase gene-deficient mice brain using two different monoclonal antibodies (anti-HNK-1 and 412). mAb 412 recognizes both non-sulfated and sulfated HNK-1 carbohydrates, as does antibody M6749. They analyzed electrophysiologically perisomatic inhibitory postsynaptic currents in hippocampal slices. Whereas application of anti-HNK-1 antibody to hippocampal slices from HNK-1 sulfotransferase gene-deficient mice did not change the perisomatic inhibitory postsynaptic currents, administration of antibody 412 decreased the perisomatic inhibitory postsynaptic currents to the same extent as did anti-HNK-1 antibody and mAb 412 in those from wild-type mice (40Senn C. Kutsche M. Saghatelyan A. Bosl M.R. Lohler J. Bartsch U. Morellini F. Schachner M. Mol. Cell. Neurosci. 2002; 20: 712-729Crossref PubMed Scopus (72) Google Scholar). The reduction of the perisomatic inhibitory postsynaptic currents after application of antibody 412 in the HNK-1 sulfotransferase gene-deficient mice indicates that the absence of sulfation does not completely abolish HNK-1 carbohydrate-induced modulation of perisomatic transmission. These experimental results indicate that the glucuronic acid residue, like the sulfate group, is an important functional constituent of the HNK-1 carbohydrate. These results suggest that the non-sulfated HNK-1 carbohydrate (glucuronic acid residue) expressed on the meprin α-subunit and CD13/aminopeptidase N also plays important roles in the kidney. We are now trying to produce GlcAT-S gene-deficient mice to demonstrate and investigate the functional role of the glucuronic acid residue of the HNK-1 carbohydrate, especially in the kidney. IntroductionGlycosylation is one of the major post-translational protein modifications that play important roles in a variety of cellular functions, including recognition and adhesion. We have been interested in the HNK-1 (human natural killer-1) carbohydrate, which is recognized by anti-HNK-1 monoclonal antibody (1Abo T. Balch C.M. J. Immunol. 1981; 127: 1024-1029PubMed Google Scholar). The carbohydrate is expressed predominantly in the nervous system in a wide range of species, and its expression is spatially and temporally regulated during development of the nervous system (2Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (141) Google Scholar, 3Yoshihara Y. Oka S. Watanabe Y. Mori K. J. Cell Biol. 1991; 115: 731-744Crossref PubMed Scopus (57) Google Scholar). The characteristic structural feature of the carbohydrate is a sulfated trisaccharide, HSO3-3GlcAβ1-3Galβ1-4GlcNAc 1The abbreviations used are: GlcA, glucuronic acid; GlcAT, glucuronyltransferase; mAb, monoclonal antibody; AQP, aquaporin; pAb, polyclonal antibody; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MSn, multistage tandem mass spectrometry. (4Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 5Voshol H. van Zuylen C.W. Orberger G. Vliegenthart J.F. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and the inner structure, Galβ1-4GlcNAc, is commonly found on various glycoproteins and glycolipids. Therefore, glucuronyltransferase(s) and sulfotransferase(s) are supposed to be key enzymes for the biosynthesis of this carbohydrate (6Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) that are involved in the biosynthesis of the HNK-1 carbohydrate from rat, mouse, and human (7Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 8Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar, 9Yamamoto S. Oka S. Saito-Ohara F. Inazawa J. Kawasaki T. J. Biochem. (Tokyo). 2002; 131: 337-347Crossref PubMed Scopus (21) Google Scholar, 10Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 65: 166-173Crossref PubMed Scopus (45) Google Scholar, 11Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To elucidate the function of the HNK-1 carbohydrate, we generated mice with a targeted deletion of the GlcAT-P gene (12Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The HNK-1 carbohydrate has disappeared almost completely in the nervous system of GlcAT-P gene-deficient mice, and these mice exhibit reduced long-term potentiation at the Schaffer collateral CA1 synapses and defects in spatial memory formation. However, a trace of anti-HNK-1 antibody immunoreactivity remains on the surfaces of the soma and proximal dendrites of a subset of neurons in some limited regions. The remaining HNK-1 carbohydrate in GlcAT-P gene-deficient mice is assumed to be synthesized by GlcAT-S. More recently, we characterized the acceptor specificities of the two glucuronyltransferases using various oligosaccharides (13Kakuda S. Sato Y. Tonoyama Y. Oka S. Kawasaki T. Glycobiology. 2005; 15: 203-210Crossref PubMed Scopus (26) Google Scholar). The results suggested the possibility that the two glucuronyltransferases synthesize structurally and functionally different HNK-1 carbohydrates.To elucidate the function of the HNK-1 carbohydrate synthesized by GlcAT-S, we examined GlcAT-S mRNA expression in various mouse tissues, including brain. To our surprise, Glc-AT-S mRNA was expressed at higher levels in the kidney than in the brain. In this study, we provide the first evidence that the HNK-1 carbohydrate is synthesized by GlcAT-S as the non-sulfated form in mouse kidney. The non-sulfated form of HNK-1 carbohydrate was expressed predominantly on meprin α and CD13/aminopeptidase N. We also examined in detail the distribution of the carbohydrate in mouse kidney. The results are important for understanding the functions of the HNK-1 carbohydrate other than those in the nervous system.
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