A Novel Endo-β-galactosidase from Clostridium perfringens That Liberates the Disaccharide GlcNAcα1→4Gal from Glycans Specifically Expressed in the Gastric Gland Mucous Cell-type Mucin
2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês
10.1074/jbc.m103589200
ISSN1083-351X
AutoresHisashi Ashida, Kimberly M. Anderson, Jun Nakayama, Karol Maskos, Chau-Wen Chou, Richard B. Cole, Su‐Chen Li, Yu‐Teh Li,
Tópico(s)Enzyme Production and Characterization
ResumoWe found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcα1→4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-α-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcα1→4Gal. The specificity of this enzyme as an endo-β-galactosidase was established by analyzing the liberation of GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol by mass spectrometry. Because this novel endo-β-galactosidase specifically releases the GlcNAcα1→4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa). Endo-β-GalGnGa was found to remove the GlcNAcα1→4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with α1,4-N-acetylglucosaminyltransferase cDNA. Endo-β-GalGnGa should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcα1→4Gal epitope. We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcα1→4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-α-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcα1→4Gal. The specificity of this enzyme as an endo-β-galactosidase was established by analyzing the liberation of GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol by mass spectrometry. Because this novel endo-β-galactosidase specifically releases the GlcNAcα1→4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa). Endo-β-GalGnGa was found to remove the GlcNAcα1→4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with α1,4-N-acetylglucosaminyltransferase cDNA. Endo-β-GalGnGa should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcα1→4Gal epitope. porcine gastric mucin GlcNAcα1→4Gal-releasing endo-β-galactosidase β-N-acetylhexosaminidase monoclonal antibody N-acetyllactosamine high performance liquid chromatography homonuclear shift correlated spectroscopy electrospray ionization-mass spectrometry α1,4-N-acetylglucosaminyltransferase 1H-detected heteronuclear single-quantum coherence rotating frame Overhauser effect spectroscopy 1H-detected heteronuclear multiple-bond correlation nuclear Overhauser effect Clostridium perfringens, a ubiquitous anaerobic bacterium commonly found in the gastrointestinal tract of higher animals and in soil, has been known to cause a wide variety of diseases in man and animals (1Smith L.D. Rev. Infect. Dis. 1979; 1: 254-262Crossref PubMed Scopus (85) Google Scholar, 2Niilo L. Can. Vet. J. 1980; 21: 141-148PubMed Google Scholar, 3Sterne M. Br. Vet. J. 1981; 137: 443-454Crossref PubMed Google Scholar). The pathogenesis caused by C. perfringens infection has been attributed to the toxins including extracellular enzymes produced by this organism (4Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar). In view of the fact that C. perfringens is the dominant cause of gastrointestinal infections in higher animals (5Borriello S.P. Clin. Infect. Dis. 1995; 20: S242-S250Crossref PubMed Scopus (70) Google Scholar), there is little doubt that this organism is endowed with enzymes capable of degrading gastrointestinal mucous glycoproteins. Although several glycosidases from C. perfringens have been detected and studied (4Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar, 6Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar), none has been shown to cleave sugar chains found in a specific class of gastrointestinal mucin. While using sialidases for structural analyses of sialoglycoconjugates, we found that commercially available sialidases prepared from C. perfringens ATCC10543 were contaminated with an unusual endoglycosidase capable of releasing a disaccharide, GlcNAcα1→4Gal, from porcine gastric mucin (PGM).1 The mucous glycoproteins containing this unique terminal disaccharide are specifically secreted from the mucous neck cell and pyloric gland of the stomach and Brunner's gland of the duodenum (7Ishihara K. Kurihara M. Goso Y. Urata T. Ota H. Katsuyama T. Hotta K. Biochem. J. 1996; 318: 409-416Crossref PubMed Scopus (129) Google Scholar, 8Nakayama J. Yeh J.C. Misra A.K. Ito S. Katsuyama T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8991-8996Crossref PubMed Scopus (86) Google Scholar). Thus, this mucin is also called the gastric gland mucous cell-type mucin. This report describes the isolation and characterization of this unusual endo-β-galactosidase from C. perfringens ATCC10543. Because this clostridial endo-β-galactosidase specifically releases the disaccharide GlcNAcα1→4Gal from PGM, we propose to call this endoglycosidase, a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa).DISCUSSIONCommercially available sialidases have been widely usedin vitro and in vivo to study the effect of desialylation of glycoconjugates. To ensure that changes after sialidase treatment are truly attributed to the removal of sialic acids, the sialidase used should be free from other contaminants. This work was initiated after an observation that commercially available sialidases prepared from C. perfringens ATCC10543 (12Cassidy J.T. Jourdian G.W. Roseman S. J. Biol. Chem. 1965; 240: 3501-3506Abstract Full Text PDF PubMed Google Scholar) were contaminated with an endoglycosidase capable of releasing a disaccharide from PGM. We have purified and characterized this unique endoglycosidase from the culture supernatant of C. perfringens. We found that this enzyme was retained by a Sephacryl S-200 HR column and could be released from the column using methyl-α-Glc. This step provided an effective one-step purification of this enzyme in electrophoretically homogeneous form. Using NMR spectroscopy (Figs. 2 B and 3 and Table II) and mass spectrometry (Fig. 4 B), we have shown unequivocally that this enzyme released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→4Galβ1→ 3)GalNAc-ol by specifically cleaving the internal Galβ1→4GlcNAc linkage in this hexasaccharide-alditol. Thus, this enzyme is an endo-β-galactosidase. However, the enzyme was not able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GalNAc-ol branch in the hexasaccharide-alditol. Using anti-Tn antigen mAb, we were able to show indirectly that the enzyme also released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→3GalNAcα1→Ser/Thr in PGM (Fig. 5). This may suggest that the pyranose structure of the aglycon, GalNAc, is important for the enzyme. Whether or not the enzyme is able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GlcNAc sequence remains to be elucidated. Oligosaccharides containing this structure are currently not available.Three different types of microbial endo-β-galactosidase have been reported. They are: (i) the endo-β-galactosidase that cleaves the endo-β-galactosyl linkages in polylactosaminoglycans (23Fukuda M.N. Matsumura G. J. Biol. Chem. 1976; 251: 6218-6225Abstract Full Text PDF PubMed Google Scholar, 24Nakagawa H. Yamada T. Chien J.L. Gardas A. Kitamikado M. Li S.-C. Li Y.-T. J. Biol. Chem. 1980; 255: 5955-5959Abstract Full Text PDF PubMed Google Scholar, 25Kitamikado M. Ito M. Li Y.-T. J. Biol. Chem. 1981; 256: 3906-3909Abstract Full Text PDF PubMed Google Scholar, 26Leng L. Zhu A. Zhang Z. Hurst R. Goldstein J. Gene (Amst.). 1998; 222: 187-194Crossref PubMed Scopus (15) Google Scholar, 27Scudder P. Uemura K. Dolby J. Fukuda M.N. Feizi T. Biochem. J. 1983; 213: 485-494Crossref PubMed Scopus (58) Google Scholar); (ii) the endo-β-galactosidase that releases blood group A and B trisaccharides from blood group A and B substances (28Takasaki S. Kobata A. J. Biol. Chem. 1976; 251: 3603-3609Abstract Full Text PDF PubMed Google Scholar); and (iii) the endo-β-galactosidase (Endo-Gal-C) that releases the Galα1→3Gal from the xenoantigen Galα1→3Galβ1→R (29Ogawa H. Muramatsu H. Kobayashi T. Morozumi K. Yokoyama I. Kurosawa N. Nakao A. Muramatsu T. J. Biol. Chem. 2000; 275: 19368-19374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The endo-β-galactosidase presented in this report is distinct from the aforementioned three endo-β-galactosidases. Because this novel endo-β-galactosidase specifically releases GlcNAcα1→4Gal from PGM, we propose to call this clostridial endoglycosidase a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa).Although the β-linked GlcNAc is found commonly in glycoconjugates, the α-linked GlcNAc is relatively rare. It is well known that glycan chains of heparin and heparan sulfate contain α-linked GlcNAc. The attachment of an α-linked GlcNAc to the threonine residues of cell surface proteins in Trypanosoma cruzi (30Previato J.O. Sola-Penna M. Agrellos O.A. Jones C. Oeltmann T. Travassos L.R. Mendonca-Previato L. J. Biol. Chem. 1998; 273: 14982-14988Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and Dictyostelium discoideum (31Jung E. Gooley A.A. Packer N.H. Karuso P. Williams K.L. Eur. J. Biochem. 1998; 253: 517-524Crossref PubMed Scopus (22) Google Scholar) has been reported recently. Lloyd et al. (32Lloyd K.O. Kabat E.A. Beychok S. J. Immunol. 1969; 102: 1354-1362PubMed Google Scholar) first reported the occurrence of the α-linked GlcNAc at the nonreducing end ofO-glycans isolated from hog gastric mucin. Later, Kochetkovet al. (33Kochetkov N.K. Derevitskaya V.A. Arbatsky N.P. Eur. J. Biochem. 1976; 67: 129-136Crossref PubMed Scopus (43) Google Scholar) isolated glycans carrying GlcNAcα1→4Gal-epitope from PGM. Van Halbeek et al. (34Van Halbeek H. Gerwig G.J. Vliegenthart J.F. Smits H.L. Van Kerkhof P.J. Kramer M.F. Biochim. Biophys. Acta. 1983; 747: 107-116Crossref PubMed Scopus (31) Google Scholar) also found the presence of this disaccharide epitope in rat duodenal gland mucin. Ishihara et al. (7Ishihara K. Kurihara M. Goso Y. Urata T. Ota H. Katsuyama T. Hotta K. Biochem. J. 1996; 318: 409-416Crossref PubMed Scopus (129) Google Scholar) developed a mAb (HIK1083) that recognizes the terminal α-GlcNAc linked to the C4 of the Gal residues in the core 2 branched O-glycan, GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol.Paradoxical concanavalin A staining, a sequential histochemical method involving periodate oxidation, sodium borohydride reduction, ConA binding, and horseradish peroxidase reaction, has been used to identify mucosubstances called class III mucin (35Katsuyama T. Spicer S.S. J. Histochem. Cytochem. 1978; 26: 233-250Crossref PubMed Scopus (293) Google Scholar). In man, the occurrence of this mucin was found to be exclusively limited to the gastric gland mucous cells, such as mucous neck and pyloric gland cells, Brunner's gland of the duodenum, and accessory gland of the pancreaticobiliary tract. Thus, class III mucin is also termed gastric gland mucous cell-type mucin. Immunohistochemical analysis of the human alimentary tract indicates that HIK1083 mAb specifically reacts with class III mucin, suggesting that this mucin contains terminally linked GlcNAcα1→4Gal residues (36Ota H. Nakayama J. Momose M. Kurihara M. Ishihara K. Hotta K. Katsuyama T. Histochem. Cell Biol. 1998; 110: 113-119Crossref PubMed Scopus (39) Google Scholar, 37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar). By using a cDNA encoding α4GnT which is responsible for the biosynthesis of GlcNAcα1→4Gal-epitope, Nakayama et al. (8Nakayama J. Yeh J.C. Misra A.K. Ito S. Katsuyama T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8991-8996Crossref PubMed Scopus (86) Google Scholar) have also successfully shown the presence of the terminal α1,4-linked GlcNAc in class III mucin. Class III mucin has also been found in gastric adenocarcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), pancreatic ductal carcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), mucinous bronchioalveolar cell carcinoma of the lung (38Honda T. Ota H. Ishii K. Nakamura N. Kubo K. Katsuyama T. Am. J. Clin. Pathol. 1998; 109: 423-430Crossref PubMed Scopus (26) Google Scholar), and the adenoma malignum of the uterine cervix (39Ishii K. Hosaka N. Toki T. Momose M. Hidaka E. Tsuchiya S. Katsuyama T. Virchows Arch. 1998; 432: 315-322Crossref PubMed Scopus (111) Google Scholar). Endo-β-GalGnGa was found to remove the GlcNAcα1→4Gal epitope expressed on the cell surface of gastric adenocarcinoma AGS-α4GnT cells (Fig. 6). The physiological consequence of releasing the GlcNAcα1→4Gal disaccharide from gastric mucin is not known at the present time. Because the GlcNAcα1→4Gal-epitope is expressed only in the gastroduodenal mucin, the removal of this disaccharide from the mucin may facilitate the passage of C. perfringens from the stomach to the intestine, the primary site of infection by this bacterium. This hypothesis can be tested in the future by comparing the pathogenicity of the wild type C. perfringens with that of the mutant devoid of Endo-β-GalGnGa activity.The existence of such an unusual endo-β-galactosidase in C. perfringens ATCC10543 is very intriguing. This unique endo-β-galactosidase should become useful for studying the structure and biological function of glycoconjugates containing the GlcNAcα1→4Gal epitope. Clostridium perfringens, a ubiquitous anaerobic bacterium commonly found in the gastrointestinal tract of higher animals and in soil, has been known to cause a wide variety of diseases in man and animals (1Smith L.D. Rev. Infect. Dis. 1979; 1: 254-262Crossref PubMed Scopus (85) Google Scholar, 2Niilo L. Can. Vet. J. 1980; 21: 141-148PubMed Google Scholar, 3Sterne M. Br. Vet. J. 1981; 137: 443-454Crossref PubMed Google Scholar). The pathogenesis caused by C. perfringens infection has been attributed to the toxins including extracellular enzymes produced by this organism (4Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar). In view of the fact that C. perfringens is the dominant cause of gastrointestinal infections in higher animals (5Borriello S.P. Clin. Infect. Dis. 1995; 20: S242-S250Crossref PubMed Scopus (70) Google Scholar), there is little doubt that this organism is endowed with enzymes capable of degrading gastrointestinal mucous glycoproteins. Although several glycosidases from C. perfringens have been detected and studied (4Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar, 6Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar), none has been shown to cleave sugar chains found in a specific class of gastrointestinal mucin. While using sialidases for structural analyses of sialoglycoconjugates, we found that commercially available sialidases prepared from C. perfringens ATCC10543 were contaminated with an unusual endoglycosidase capable of releasing a disaccharide, GlcNAcα1→4Gal, from porcine gastric mucin (PGM).1 The mucous glycoproteins containing this unique terminal disaccharide are specifically secreted from the mucous neck cell and pyloric gland of the stomach and Brunner's gland of the duodenum (7Ishihara K. Kurihara M. Goso Y. Urata T. Ota H. Katsuyama T. Hotta K. Biochem. J. 1996; 318: 409-416Crossref PubMed Scopus (129) Google Scholar, 8Nakayama J. Yeh J.C. Misra A.K. Ito S. Katsuyama T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8991-8996Crossref PubMed Scopus (86) Google Scholar). Thus, this mucin is also called the gastric gland mucous cell-type mucin. This report describes the isolation and characterization of this unusual endo-β-galactosidase from C. perfringens ATCC10543. Because this clostridial endo-β-galactosidase specifically releases the disaccharide GlcNAcα1→4Gal from PGM, we propose to call this endoglycosidase, a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa). DISCUSSIONCommercially available sialidases have been widely usedin vitro and in vivo to study the effect of desialylation of glycoconjugates. To ensure that changes after sialidase treatment are truly attributed to the removal of sialic acids, the sialidase used should be free from other contaminants. This work was initiated after an observation that commercially available sialidases prepared from C. perfringens ATCC10543 (12Cassidy J.T. Jourdian G.W. Roseman S. J. Biol. Chem. 1965; 240: 3501-3506Abstract Full Text PDF PubMed Google Scholar) were contaminated with an endoglycosidase capable of releasing a disaccharide from PGM. We have purified and characterized this unique endoglycosidase from the culture supernatant of C. perfringens. We found that this enzyme was retained by a Sephacryl S-200 HR column and could be released from the column using methyl-α-Glc. This step provided an effective one-step purification of this enzyme in electrophoretically homogeneous form. Using NMR spectroscopy (Figs. 2 B and 3 and Table II) and mass spectrometry (Fig. 4 B), we have shown unequivocally that this enzyme released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→4Galβ1→ 3)GalNAc-ol by specifically cleaving the internal Galβ1→4GlcNAc linkage in this hexasaccharide-alditol. Thus, this enzyme is an endo-β-galactosidase. However, the enzyme was not able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GalNAc-ol branch in the hexasaccharide-alditol. Using anti-Tn antigen mAb, we were able to show indirectly that the enzyme also released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→3GalNAcα1→Ser/Thr in PGM (Fig. 5). This may suggest that the pyranose structure of the aglycon, GalNAc, is important for the enzyme. Whether or not the enzyme is able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GlcNAc sequence remains to be elucidated. Oligosaccharides containing this structure are currently not available.Three different types of microbial endo-β-galactosidase have been reported. They are: (i) the endo-β-galactosidase that cleaves the endo-β-galactosyl linkages in polylactosaminoglycans (23Fukuda M.N. Matsumura G. J. Biol. Chem. 1976; 251: 6218-6225Abstract Full Text PDF PubMed Google Scholar, 24Nakagawa H. Yamada T. Chien J.L. Gardas A. Kitamikado M. Li S.-C. Li Y.-T. J. Biol. Chem. 1980; 255: 5955-5959Abstract Full Text PDF PubMed Google Scholar, 25Kitamikado M. Ito M. Li Y.-T. J. Biol. Chem. 1981; 256: 3906-3909Abstract Full Text PDF PubMed Google Scholar, 26Leng L. Zhu A. Zhang Z. Hurst R. Goldstein J. Gene (Amst.). 1998; 222: 187-194Crossref PubMed Scopus (15) Google Scholar, 27Scudder P. Uemura K. Dolby J. Fukuda M.N. Feizi T. Biochem. J. 1983; 213: 485-494Crossref PubMed Scopus (58) Google Scholar); (ii) the endo-β-galactosidase that releases blood group A and B trisaccharides from blood group A and B substances (28Takasaki S. Kobata A. J. Biol. Chem. 1976; 251: 3603-3609Abstract Full Text PDF PubMed Google Scholar); and (iii) the endo-β-galactosidase (Endo-Gal-C) that releases the Galα1→3Gal from the xenoantigen Galα1→3Galβ1→R (29Ogawa H. Muramatsu H. Kobayashi T. Morozumi K. Yokoyama I. Kurosawa N. Nakao A. Muramatsu T. J. Biol. Chem. 2000; 275: 19368-19374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The endo-β-galactosidase presented in this report is distinct from the aforementioned three endo-β-galactosidases. Because this novel endo-β-galactosidase specifically releases GlcNAcα1→4Gal from PGM, we propose to call this clostridial endoglycosidase a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa).Although the β-linked GlcNAc is found commonly in glycoconjugates, the α-linked GlcNAc is relatively rare. It is well known that glycan chains of heparin and heparan sulfate contain α-linked GlcNAc. The attachment of an α-linked GlcNAc to the threonine residues of cell surface proteins in Trypanosoma cruzi (30Previato J.O. Sola-Penna M. Agrellos O.A. Jones C. Oeltmann T. Travassos L.R. Mendonca-Previato L. J. Biol. Chem. 1998; 273: 14982-14988Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and Dictyostelium discoideum (31Jung E. Gooley A.A. Packer N.H. Karuso P. Williams K.L. Eur. J. Biochem. 1998; 253: 517-524Crossref PubMed Scopus (22) Google Scholar) has been reported recently. Lloyd et al. (32Lloyd K.O. Kabat E.A. Beychok S. J. Immunol. 1969; 102: 1354-1362PubMed Google Scholar) first reported the occurrence of the α-linked GlcNAc at the nonreducing end ofO-glycans isolated from hog gastric mucin. Later, Kochetkovet al. (33Kochetkov N.K. Derevitskaya V.A. Arbatsky N.P. Eur. J. Biochem. 1976; 67: 129-136Crossref PubMed Scopus (43) Google Scholar) isolated glycans carrying GlcNAcα1→4Gal-epitope from PGM. Van Halbeek et al. (34Van Halbeek H. Gerwig G.J. Vliegenthart J.F. Smits H.L. Van Kerkhof P.J. Kramer M.F. Biochim. Biophys. Acta. 1983; 747: 107-116Crossref PubMed Scopus (31) Google Scholar) also found the presence of this disaccharide epitope in rat duodenal gland mucin. Ishihara et al. (7Ishihara K. Kurihara M. Goso Y. Urata T. Ota H. Katsuyama T. Hotta K. Biochem. J. 1996; 318: 409-416Crossref PubMed Scopus (129) Google Scholar) developed a mAb (HIK1083) that recognizes the terminal α-GlcNAc linked to the C4 of the Gal residues in the core 2 branched O-glycan, GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol.Paradoxical concanavalin A staining, a sequential histochemical method involving periodate oxidation, sodium borohydride reduction, ConA binding, and horseradish peroxidase reaction, has been used to identify mucosubstances called class III mucin (35Katsuyama T. Spicer S.S. J. Histochem. Cytochem. 1978; 26: 233-250Crossref PubMed Scopus (293) Google Scholar). In man, the occurrence of this mucin was found to be exclusively limited to the gastric gland mucous cells, such as mucous neck and pyloric gland cells, Brunner's gland of the duodenum, and accessory gland of the pancreaticobiliary tract. Thus, class III mucin is also termed gastric gland mucous cell-type mucin. Immunohistochemical analysis of the human alimentary tract indicates that HIK1083 mAb specifically reacts with class III mucin, suggesting that this mucin contains terminally linked GlcNAcα1→4Gal residues (36Ota H. Nakayama J. Momose M. Kurihara M. Ishihara K. Hotta K. Katsuyama T. Histochem. Cell Biol. 1998; 110: 113-119Crossref PubMed Scopus (39) Google Scholar, 37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar). By using a cDNA encoding α4GnT which is responsible for the biosynthesis of GlcNAcα1→4Gal-epitope, Nakayama et al. (8Nakayama J. Yeh J.C. Misra A.K. Ito S. Katsuyama T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8991-8996Crossref PubMed Scopus (86) Google Scholar) have also successfully shown the presence of the terminal α1,4-linked GlcNAc in class III mucin. Class III mucin has also been found in gastric adenocarcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), pancreatic ductal carcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), mucinous bronchioalveolar cell carcinoma of the lung (38Honda T. Ota H. Ishii K. Nakamura N. Kubo K. Katsuyama T. Am. J. Clin. Pathol. 1998; 109: 423-430Crossref PubMed Scopus (26) Google Scholar), and the adenoma malignum of the uterine cervix (39Ishii K. Hosaka N. Toki T. Momose M. Hidaka E. Tsuchiya S. Katsuyama T. Virchows Arch. 1998; 432: 315-322Crossref PubMed Scopus (111) Google Scholar). Endo-β-GalGnGa was found to remove the GlcNAcα1→4Gal epitope expressed on the cell surface of gastric adenocarcinoma AGS-α4GnT cells (Fig. 6). The physiological consequence of releasing the GlcNAcα1→4Gal disaccharide from gastric mucin is not known at the present time. Because the GlcNAcα1→4Gal-epitope is expressed only in the gastroduodenal mucin, the removal of this disaccharide from the mucin may facilitate the passage of C. perfringens from the stomach to the intestine, the primary site of infection by this bacterium. This hypothesis can be tested in the future by comparing the pathogenicity of the wild type C. perfringens with that of the mutant devoid of Endo-β-GalGnGa activity.The existence of such an unusual endo-β-galactosidase in C. perfringens ATCC10543 is very intriguing. This unique endo-β-galactosidase should become useful for studying the structure and biological function of glycoconjugates containing the GlcNAcα1→4Gal epitope. Commercially available sialidases have been widely usedin vitro and in vivo to study the effect of desialylation of glycoconjugates. To ensure that changes after sialidase treatment are truly attributed to the removal of sialic acids, the sialidase used should be free from other contaminants. This work was initiated after an observation that commercially available sialidases prepared from C. perfringens ATCC10543 (12Cassidy J.T. Jourdian G.W. Roseman S. J. Biol. Chem. 1965; 240: 3501-3506Abstract Full Text PDF PubMed Google Scholar) were contaminated with an endoglycosidase capable of releasing a disaccharide from PGM. We have purified and characterized this unique endoglycosidase from the culture supernatant of C. perfringens. We found that this enzyme was retained by a Sephacryl S-200 HR column and could be released from the column using methyl-α-Glc. This step provided an effective one-step purification of this enzyme in electrophoretically homogeneous form. Using NMR spectroscopy (Figs. 2 B and 3 and Table II) and mass spectrometry (Fig. 4 B), we have shown unequivocally that this enzyme released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→4Galβ1→ 3)GalNAc-ol by specifically cleaving the internal Galβ1→4GlcNAc linkage in this hexasaccharide-alditol. Thus, this enzyme is an endo-β-galactosidase. However, the enzyme was not able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GalNAc-ol branch in the hexasaccharide-alditol. Using anti-Tn antigen mAb, we were able to show indirectly that the enzyme also released GlcNAcα1→4Gal from GlcNAcα1→4Galβ1→3GalNAcα1→Ser/Thr in PGM (Fig. 5). This may suggest that the pyranose structure of the aglycon, GalNAc, is important for the enzyme. Whether or not the enzyme is able to release GlcNAcα1→4Gal from the GlcNAcα1→4Galβ1→3GlcNAc sequence remains to be elucidated. Oligosaccharides containing this structure are currently not available. Three different types of microbial endo-β-galactosidase have been reported. They are: (i) the endo-β-galactosidase that cleaves the endo-β-galactosyl linkages in polylactosaminoglycans (23Fukuda M.N. Matsumura G. J. Biol. Chem. 1976; 251: 6218-6225Abstract Full Text PDF PubMed Google Scholar, 24Nakagawa H. Yamada T. Chien J.L. Gardas A. Kitamikado M. Li S.-C. Li Y.-T. J. Biol. Chem. 1980; 255: 5955-5959Abstract Full Text PDF PubMed Google Scholar, 25Kitamikado M. Ito M. Li Y.-T. J. Biol. Chem. 1981; 256: 3906-3909Abstract Full Text PDF PubMed Google Scholar, 26Leng L. Zhu A. Zhang Z. Hurst R. Goldstein J. Gene (Amst.). 1998; 222: 187-194Crossref PubMed Scopus (15) Google Scholar, 27Scudder P. Uemura K. Dolby J. Fukuda M.N. Feizi T. Biochem. J. 1983; 213: 485-494Crossref PubMed Scopus (58) Google Scholar); (ii) the endo-β-galactosidase that releases blood group A and B trisaccharides from blood group A and B substances (28Takasaki S. Kobata A. J. Biol. Chem. 1976; 251: 3603-3609Abstract Full Text PDF PubMed Google Scholar); and (iii) the endo-β-galactosidase (Endo-Gal-C) that releases the Galα1→3Gal from the xenoantigen Galα1→3Galβ1→R (29Ogawa H. Muramatsu H. Kobayashi T. Morozumi K. Yokoyama I. Kurosawa N. Nakao A. Muramatsu T. J. Biol. Chem. 2000; 275: 19368-19374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The endo-β-galactosidase presented in this report is distinct from the aforementioned three endo-β-galactosidases. Because this novel endo-β-galactosidase specifically releases GlcNAcα1→4Gal from PGM, we propose to call this clostridial endoglycosidase a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa). Although the β-linked GlcNAc is found commonly in glycoconjugates, the α-linked GlcNAc is relatively rare. It is well known that glycan chains of heparin and heparan sulfate contain α-linked GlcNAc. The attachment of an α-linked GlcNAc to the threonine residues of cell surface proteins in Trypanosoma cruzi (30Previato J.O. Sola-Penna M. Agrellos O.A. Jones C. Oeltmann T. Travassos L.R. Mendonca-Previato L. J. Biol. Chem. 1998; 273: 14982-14988Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and Dictyostelium discoideum (31Jung E. Gooley A.A. Packer N.H. Karuso P. Williams K.L. Eur. J. Biochem. 1998; 253: 517-524Crossref PubMed Scopus (22) Google Scholar) has been reported recently. Lloyd et al. (32Lloyd K.O. Kabat E.A. Beychok S. J. Immunol. 1969; 102: 1354-1362PubMed Google Scholar) first reported the occurrence of the α-linked GlcNAc at the nonreducing end ofO-glycans isolated from hog gastric mucin. Later, Kochetkovet al. (33Kochetkov N.K. Derevitskaya V.A. Arbatsky N.P. Eur. J. Biochem. 1976; 67: 129-136Crossref PubMed Scopus (43) Google Scholar) isolated glycans carrying GlcNAcα1→4Gal-epitope from PGM. Van Halbeek et al. (34Van Halbeek H. Gerwig G.J. Vliegenthart J.F. Smits H.L. Van Kerkhof P.J. Kramer M.F. Biochim. Biophys. Acta. 1983; 747: 107-116Crossref PubMed Scopus (31) Google Scholar) also found the presence of this disaccharide epitope in rat duodenal gland mucin. Ishihara et al. (7Ishihara K. Kurihara M. Goso Y. Urata T. Ota H. Katsuyama T. Hotta K. Biochem. J. 1996; 318: 409-416Crossref PubMed Scopus (129) Google Scholar) developed a mAb (HIK1083) that recognizes the terminal α-GlcNAc linked to the C4 of the Gal residues in the core 2 branched O-glycan, GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol. Paradoxical concanavalin A staining, a sequential histochemical method involving periodate oxidation, sodium borohydride reduction, ConA binding, and horseradish peroxidase reaction, has been used to identify mucosubstances called class III mucin (35Katsuyama T. Spicer S.S. J. Histochem. Cytochem. 1978; 26: 233-250Crossref PubMed Scopus (293) Google Scholar). In man, the occurrence of this mucin was found to be exclusively limited to the gastric gland mucous cells, such as mucous neck and pyloric gland cells, Brunner's gland of the duodenum, and accessory gland of the pancreaticobiliary tract. Thus, class III mucin is also termed gastric gland mucous cell-type mucin. Immunohistochemical analysis of the human alimentary tract indicates that HIK1083 mAb specifically reacts with class III mucin, suggesting that this mucin contains terminally linked GlcNAcα1→4Gal residues (36Ota H. Nakayama J. Momose M. Kurihara M. Ishihara K. Hotta K. Katsuyama T. Histochem. Cell Biol. 1998; 110: 113-119Crossref PubMed Scopus (39) Google Scholar, 37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar). By using a cDNA encoding α4GnT which is responsible for the biosynthesis of GlcNAcα1→4Gal-epitope, Nakayama et al. (8Nakayama J. Yeh J.C. Misra A.K. Ito S. Katsuyama T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8991-8996Crossref PubMed Scopus (86) Google Scholar) have also successfully shown the presence of the terminal α1,4-linked GlcNAc in class III mucin. Class III mucin has also been found in gastric adenocarcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), pancreatic ductal carcinoma (37Nakamura N. Ota H. Katsuyama T. Akamatsu T. Ishihara K. Kurihara M. Hotta K. J. Histochem. Cytochem. 1998; 46: 793-801Crossref PubMed Scopus (62) Google Scholar), mucinous bronchioalveolar cell carcinoma of the lung (38Honda T. Ota H. Ishii K. Nakamura N. Kubo K. Katsuyama T. Am. J. Clin. Pathol. 1998; 109: 423-430Crossref PubMed Scopus (26) Google Scholar), and the adenoma malignum of the uterine cervix (39Ishii K. Hosaka N. Toki T. Momose M. Hidaka E. Tsuchiya S. Katsuyama T. Virchows Arch. 1998; 432: 315-322Crossref PubMed Scopus (111) Google Scholar). Endo-β-GalGnGa was found to remove the GlcNAcα1→4Gal epitope expressed on the cell surface of gastric adenocarcinoma AGS-α4GnT cells (Fig. 6). The physiological consequence of releasing the GlcNAcα1→4Gal disaccharide from gastric mucin is not known at the present time. Because the GlcNAcα1→4Gal-epitope is expressed only in the gastroduodenal mucin, the removal of this disaccharide from the mucin may facilitate the passage of C. perfringens from the stomach to the intestine, the primary site of infection by this bacterium. This hypothesis can be tested in the future by comparing the pathogenicity of the wild type C. perfringens with that of the mutant devoid of Endo-β-GalGnGa activity. The existence of such an unusual endo-β-galactosidase in C. perfringens ATCC10543 is very intriguing. This unique endo-β-galactosidase should become useful for studying the structure and biological function of glycoconjugates containing the GlcNAcα1→4Gal epitope.
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