Molecular Cloning and Characterization of a Plasma Membrane-associated Sialidase Specific for Gangliosides

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19–38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both α2→3 and α2→8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence. Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19–38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both α2→3 and α2→8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence. The sialidase reaction is an initial step of the degradation of glycoproteins and gangliosides. Sialidases of mammalian origin have been implicated not only in lysosomal catabolism but also in modulation of functional molecules involved in many biological processes (1Schauer R. Trends Biochem. Sci. 1985; 10: 357-360Abstract Full Text PDF Scopus (485) Google Scholar, 2Pilatte Y. Bignon J. Lambré C.R. Glycobiology. 1993; 3: 201-217Crossref PubMed Scopus (152) Google Scholar). However, the physiological significance and the regulation mechanisms of desialylation remain obscure because the structure and function of mammalian sialidases are not fully understood. Our previous studies aimed at the biochemical characterization of mammalian sialidases demonstrated four types in rat tissues differing in subcellular location as well as catalytic and immunological properties: intralysosomal (3Miyagi T. Tsuiki S. Eur. J Biochem. 1984; 141: 75-81Crossref PubMed Scopus (106) Google Scholar), cytosolic (4Miyagi T. Tsuiki S. J. Biol. Chem. 1985; 260: 6710-6716Abstract Full Text PDF PubMed Google Scholar), lysosomal membrane, and plasma membrane (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar). The multiple nature of mammalian sialidases suggests that each form may play a unique role depending on its particular subcellular location and catalytic properties. To elucidate the structure and function of these low abundance proteins, cloning of the individual genes is required. We previously cloned a rat cytosolic sialidase gene (6Miyagi T. Konno K. Emori Y. Kawasaki H. Suzuki K. Yasui A. Tsuiki S. J. Biol. Chem. 1993; 268: 26435-26440Abstract Full Text PDF PubMed Google Scholar), the first cDNA example of a mammalian species, and established its involvement in differentiation of skeletal muscle cells (7Sato K. Miyagi T. Biochem. Biophys. Res. Commun. 1996; 221: 826-830Crossref PubMed Scopus (65) Google Scholar). Recently, human (8Bonten E. van der Spoel A. Fornerod M. Grosveld G. d'Azoo A. Genes Dev. 1996; 10: 3156-3169Crossref PubMed Scopus (252) Google Scholar, 9Milner C.M. Smith S.V. Carrillo M.B. Taylor G.L. Hollinshead M. Campbell R.D. J. Biol. Chem. 1997; 272: 4549-4558Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Pshezhetsky A.V. Richard C. Michaud L. Igdoura S. Wang S. Elsliger M.-A. Qu J. Leclerc D. Gravel R. Dallaire L. Potier M. Nat. Genet. 1997; 18: 316-320Crossref Scopus (182) Google Scholar) and mouse (11Carrillo M.B. Milner C.M. Ball S.T. Snoek M. Campbell R.D. Glycobiology. 1997; 7: 975-986Crossref PubMed Scopus (59) Google Scholar, 12Igdoura S.A. Gafuik C. Mertineit C. Saberi F. Pshezhetsky A.V. Potier M. Trasler J.M. Gravel R.A. Hum. Mol. Genet. 1998; 7: 115-121Crossref PubMed Scopus (56) Google Scholar) major histocompatibility complex ( MHC) 1The abbreviations MHCmajor histocompatibility complexMOPS4-morpholinepropanesulfonic acid4MU-NeuAc4-methylumbelliferyl-neuraminic acidPCRpolymerase chain reactionHSVHerpes Simplex virusaaamino acid(s)PAGEpolyacrylamide gel electrophoresis 1The abbreviations MHCmajor histocompatibility complexMOPS4-morpholinepropanesulfonic acid4MU-NeuAc4-methylumbelliferyl-neuraminic acidPCRpolymerase chain reactionHSVHerpes Simplex virusaaamino acid(s)PAGEpolyacrylamide gel electrophoresis-related sialidases were cloned and suggested to be primarily localized in lysosomes (13van der Spoel A. Bonten E. d'Azoo A. EMBO J. 1998; 17: 1588-1597Crossref PubMed Scopus (105) Google Scholar). major histocompatibility complex 4-morpholinepropanesulfonic acid 4-methylumbelliferyl-neuraminic acid polymerase chain reaction Herpes Simplex virus amino acid(s) polyacrylamide gel electrophoresis major histocompatibility complex 4-morpholinepropanesulfonic acid 4-methylumbelliferyl-neuraminic acid polymerase chain reaction Herpes Simplex virus amino acid(s) polyacrylamide gel electrophoresis Membrane-associated sialidases hydrolyze gangliosides preferentially (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar, 14Öhman R. Svennerholm L. J. Neurochem. 1971; 18: 79-87Crossref PubMed Scopus (16) Google Scholar, 15Öhman R. J. Neurochem. 1971; 18: 89-95Crossref PubMed Scopus (21) Google Scholar), and those in the plasma membrane are distinct from lysosomal membrane sialidases in acting specifically on gangliosides (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar, 16Kopitz J. von Reitzenstein C. Sinz K. M. C. Glycobiology. 1996; 6: 367-376Crossref PubMed Scopus (68) Google Scholar, 17Kopitz J. Sinz K. Brossmer R. Cantz M. Eur. J. Biochem. 1997; 248: 527-534Crossref PubMed Scopus (44) Google Scholar, 18Hata K. Wada T. Hasegawa A. Kiso M. Miyagi T. J. Biochem. 1998; 123: 899-905Crossref PubMed Scopus (50) Google Scholar) residing in the same membrane. Gangliosides are thought to play crucial roles in cell surface events, including cell differentiation, cell-cell interactions, and transmembrane signaling (19Zeller C.B. Marchase R.B. Am. J. Physiol. 1992; 262: C1341-C1355Crossref PubMed Google Scholar, 20Hakomori S. Biochem. Soc. Trans. 1993; 21: 583-595Crossref PubMed Scopus (167) Google Scholar, 21Tettamanti G. Riboni L. Adv. Lipid Res. 1993; 25: 235-267PubMed Google Scholar). Plasma membrane sialidases, therefore, have been considered to participate in these phenomena through modulation of gangliosides. In fact, there are observations suggesting important biological roles of ganglioside sialidases, although information as to what types of ganglioside sialidase involved is not available enough. The activity levels fluctuate consistently with cell differentiation, cell growth, and malignant transformation. For example, a sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, abolishes increase of a differentiation marker enzyme in human neuroblastoma cells (22Kopitz J. von Reitzenstein C. Muhl C. Cantz M. Biochem. Biophys. Res. Commun. 1994; 199: 1188-1193Crossref PubMed Scopus (78) Google Scholar, 23Kopitz J. Muhl C. Ehemann V. Lehmann C. Cantz M. Eur. J. Cell Biol. 1997; 73: 1-9PubMed Google Scholar), and the observations by Usuki et al. (24Usuki S. Lyu S.-C. Sweeley C.C. J. Biol. Chem. 1988; 263: 6847-6853Abstract Full Text PDF PubMed Google Scholar,25Usuki S. Hoops P. Sweeley C.C. J. Biol. Chem. 1988; 263: 10595-10599Abstract Full Text PDF PubMed Google Scholar) led them to propose the participation of ganglioside sialidase in cell growth regulation. In addition, alterations of the levels of ganglioside sialidase expression associated with malignant transformation have been described: loss of cell density-dependent suppression in 3T3-transformed cells (26Yogeeswaran G. Hakomori S. Biochemistry. 1975; 14: 2151-2156Crossref PubMed Scopus (80) Google Scholar) and appearance of ganglioside sialidase activity in transformed cell lines of baby hamster kidney fibroblasts (27Schengrund C.-L. Lausch R.N. Rosenberg A. J. Biol. Chem. 1973; 248: 4424-4428Abstract Full Text PDF PubMed Google Scholar). We previously reported an increase of plasma membrane sialidase activity associated with induction of anchorage-independent growth in mouse epidermal JB6 cells exposed to phorbol esters (28Miyagi T. Sagawa J. Kuroki T. Matsuya Y. Tsuiki S. Jpn. J. Cancer Res. 1990; 81: 1286-1292Crossref PubMed Scopus (22) Google Scholar). However, little is known about the molecular mechanisms underlying such sialidase alterations. To provide tools for their elucidation, we have focused on cloning a cDNA of the sialidase. We recently were able to purify a ganglioside sialidase extensively from bovine brain, which is the major ganglioside-hydrolyzing sialidase of the tissue and is located mainly in synaptosomes (18Hata K. Wada T. Hasegawa A. Kiso M. Miyagi T. J. Biochem. 1998; 123: 899-905Crossref PubMed Scopus (50) Google Scholar). Using the purified enzyme protein, we have now succeeded in cloning a membrane-associated ganglioside sialidase. Membrane-associated sialidase was purified extensively from 5 kg of frozen bovine brain as described previously (18Hata K. Wada T. Hasegawa A. Kiso M. Miyagi T. J. Biochem. 1998; 123: 899-905Crossref PubMed Scopus (50) Google Scholar). The concentrated enzyme at the step of thiol-activated Sepharose column chromatography was electrophoresed on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The polyvinylidene enzyme protein was to and in with and A. J. Biochem. 1996; PubMed Scopus Google Scholar). The were on a chromatography and amino acid was on the amino acid of the four from the purified sialidase for both and were with as in were from the of bovine brain and virus and as for The were as at for at for at for A with and was to be by with and as a to a bovine brain cDNA library were isolated by and by the chain in both an and containing were and For and the described by et al. Sci. S. A. 1988; PubMed Scopus Google Scholar) was specific based on the sequence of the To sialidase cDNA of mouse and human were the same described with the and for a in which the amino acid sequence of the bovine sialidase is to that of cytosolic sialidase. was from bovine brain by the acid P. Biochem. PubMed Scopus Google Scholar), and was isolated by column from human brain and skeletal muscle were from and were at in a of and electrophoresed in a gel containing and transferred to a membrane. The membrane was with cDNA at in a containing 5 5 and and in and in at A sialidase expression was by the sequence containing the by at an at as the of the The sialidase expression was by the for the of the sialidase cDNA in the The expression was transfected COS-7 cells by of growth in cell cells were by in of containing and at and at for and the was for sialidase activity The reaction of as acid, of bovine of and of Triton at for the acid was by the acid as described (4Miyagi T. Tsuiki S. J. Biol. Chem. 1985; 260: 6710-6716Abstract Full Text PDF PubMed Google Scholar). activity 4-methylumbelliferyl-neuraminic acid was by of (4Miyagi T. Tsuiki S. J. Biol. Chem. 1985; 260: 6710-6716Abstract Full Text PDF PubMed Google Scholar). of sialidase was as the of enzyme that the of of Percoll density gradient centrifugation of the sialidase in transfected cells was as of cell was on of of Percoll with of and at of were and for sialidase, and (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar). For immunofluorescence transfected cells expressing sialidase were with for with Triton in for and with a cells transfected with in which the of membrane sialidase was by that of cytosolic sialidase (6Miyagi T. Konno K. Emori Y. Kawasaki H. Suzuki K. Yasui A. Tsuiki S. J. Biol. Chem. 1993; 268: 26435-26440Abstract Full Text PDF PubMed Google Scholar), were in the same To to the cell cells transfected with were with The was by with to amino acid to The specific was purified from the of and were as To the membrane topology of the sialidase, a protection assay was cells were in of containing and and by through a The were to with for at in the presence of Triton the reaction with 5 the was at for and the were in of sialidase were by by to The presence in the expressed sialidase was assessed by and was the at at to For homogenates of of the transfected cells were with of at as by the was as a control The enzyme protein was with by and on the four for and were and in the reaction with bovine brain cDNA as cDNA with and was as a because the amino acid sequence Asp boxes P. J. L. R. J. 6: PubMed Scopus Google Scholar) and demonstrated identity with cytosolic sialidase this cDNA as a we a bovine brain library and were found to the and the by in at the of The and the cDNA with an were to a cDNA of an of with a putative in with the for We that the at is the than the at because of the of a in the of of the were found to a signal but were in the thought to be involved in of for and and in the regulation of gene expression cell growth and differentiation Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). of the of the gene by was The protein has amino with a molecular of that the four three Asp and of the sequence with those of other mammalian and sialidases revealed 19–38% sequence the of was found with rat (6Miyagi T. Konno K. Emori Y. Kawasaki H. Suzuki K. Yasui A. Tsuiki S. J. Biol. Chem. 1993; 268: 26435-26440Abstract Full Text PDF PubMed Google Scholar) and hamster J. R. 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PubMed Scopus Google Scholar) suggested that the sialidase has a of amino and signal sequence as in The the sialidase sequence and be to be a transmembrane by a on the and sialidases, this enzyme has a of and S. sialidase, structure has been by Taylor G.L. Sci. S. A. 1993; PubMed Scopus Google Scholar), was to of bovine sialidase. of the the described by Milner et al. C.M. Smith S.V. Carrillo M.B. Taylor G.L. Hollinshead M. Campbell R.D. J. Biol. Chem. 1997; 272: 4549-4558Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) revealed a of the catalytic of the are in the bovine sialidase. of the three involved in the of acid and and three of the four the and in the demonstrated by and in the bovine The three in the are to the in the rat cytosolic sialidase, and four from those of human in Northern blot of bovine brain revealed an of the a cDNA the sequence as a a of the gene at the The same were found to be in human skeletal muscle and brain. Analysis of the by reaction for the human and mouse sialidase as in demonstrated sequence identity to the bovine In this the four the as with sialidase, were and their were In fact, the sequence of the human gene an identity not To that the isolated cDNA encodes the ganglioside-hydrolyzing sialidase, were by of the cDNA a expression containing the COS-7 cells transiently transfected with an 40-fold increase in sialidase activity gangliosides in the presence of Triton cell homogenates and as enzyme in with cells with the activity level a was not The sialidase preferentially on gangliosides other than GM1 and and on both sialyl linkages of α2→3 and but on and as demonstrated previously (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar, 23Kopitz J. Muhl C. Ehemann V. Lehmann C. Cantz M. Eur. J. Cell Biol. 1997; 73: 1-9PubMed Google Scholar) with the purified enzyme from bovine brain the transfected cell homogenates were to Percoll density gradient sialidase activity gangliosides in the presence of Triton with a plasma membrane marker but little was in the to lysosomes To the with the plasma membrane, transfected cells with sialidase genes were and with and by 5 surface staining was in cells expressing the sialidase, of the cytosolic sialidase gene in cytosolic and as demonstrated previously in skeletal muscle cells by H. Miyagi T. K. M. Cell Biol. 1997; 107: PubMed Scopus (22) Google Scholar), of the with the sialidase We the sialidase is a transmembrane protein and is an to the at the terminus and an to the to amino acid of the putative transmembrane expressing the same of sialidase activity as cells. in 5 the sialidase was specifically as a of on with 5 and and The presence of in cell a little in of the protein on but of the homogenates with in the a control its little of the presence of membrane were with and the were by with its and an form was with the and which the on the side of the sialidase, but protection was with the sialidase The by to the amino terminus and a of the domain being with in the presence of Triton This expressed sialidase, therefore, to be a transmembrane protein with an extracytoplasmic amino for this was by of transfected cells. The to a cell surface low staining not staining was The for transmembrane by the R. P. C. Sci. PubMed Scopus Google Scholar) and K. Biol. Chem. 1993; Scholar) is with these protein expression at the amino terminus demonstrated sialidase and of the with expression sialidase these that the ganglioside sialidase is associated with plasma membrane and that is an type I membrane protein lacking a signal sequence and the amino terminus to the extracytoplasmic types of mammalian sialidase, major are the (6Miyagi T. Konno K. Emori Y. Kawasaki H. Suzuki K. Yasui A. Tsuiki S. J. Biol. Chem. 1993; 268: 26435-26440Abstract Full Text PDF PubMed Google Scholar, Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar) and lysosomes (8Bonten E. van der Spoel A. Fornerod M. Grosveld G. d'Azoo A. Genes Dev. 1996; 10: 3156-3169Crossref PubMed Scopus (252) Google Scholar, 9Milner C.M. Smith S.V. Carrillo M.B. Taylor G.L. Hollinshead M. Campbell R.D. J. Biol. Chem. 1997; 272: 4549-4558Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Pshezhetsky A.V. Richard C. Michaud L. Igdoura S. Wang S. Elsliger M.-A. Qu J. Leclerc D. Gravel R. Dallaire L. Potier M. Nat. Genet. 1997; 18: 316-320Crossref Scopus (182) Google Scholar, M.B. Milner C.M. Ball S.T. Snoek M. Campbell R.D. Glycobiology. 1997; 7: 975-986Crossref PubMed Scopus (59) Google Scholar, 12Igdoura S.A. Gafuik C. Mertineit C. Saberi F. Pshezhetsky A.V. Potier M. Trasler J.M. Gravel R.A. Hum. Mol. Genet. 1998; 7: 115-121Crossref PubMed Scopus (56) Google Scholar), have been cloned to are not but including boxes characteristic of sialidases. The plasma membrane-associated sialidase has been found to be distinct from cytosolic and lysosomal sialidases in in its with hydrolysis of gangliosides but not glycoproteins to (5Miyagi T. Sagawa J. Konno K. Handa S. Tsuiki S. J. Biochem. 1990; 107: 787-793Crossref PubMed Scopus (85) Google Scholar, 17Kopitz J. Sinz K. Brossmer R. Cantz M. Eur. J. Biochem. 1997; 248: 527-534Crossref PubMed Scopus (44) Google Scholar, 18Hata K. Wada T. Hasegawa A. Kiso M. Miyagi T. J. 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Chem. 1997; 272: 4549-4558Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). the in sequence and structure that the three mammalian sialidases a with sialidases, there be for the in the being involved because of their from those of sialidase as well as sialidases. the containing these was to be on the side of the plasma membrane by protease protection for subcellular location are characteristic for each the ganglioside sialidase a putative transmembrane such for cytosolic and lysosomal sialidases, have a signal and a lysosomal sequence. COS-7 cells transiently transfected with the cDNA sialidase activity ganglioside in the presence of Triton in the This is with observations cell homogenates as the enzyme by et al. M. E. H. G. Cantz M. Biochem. J. 260: PubMed Scopus Google Scholar) that plasma membrane sialidase is by suggesting that the activity was because of plasma membrane sialidase. The that the of the expressed sialidase gangliosides was to that of the purified enzyme from bovine brain that the cDNA encodes the sialidase The subcellular localization of this sialidase was to be the plasma membrane by density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected cells. was able to in subcellular localization the ganglioside sialidase and rat cytosolic sialidase as a an of the on expression of these genes because of the with previous for specific to the cytosolic sialidase H. Miyagi T. K. M. Cell Biol. 1997; 107: PubMed Scopus (22) Google Scholar). of the transmembrane topology of the sialidase by protease protection for an extracytoplasmic side at the amino that a putative in the This a is functional on the sialidase protein expressed in COS-7 cells because previous that the sialidase at a step from bovine brain have suggested that the sialidase may be an protein (18Hata K. Wada T. Hasegawa A. Kiso M. Miyagi T. J. Biochem. 1998; 123: 899-905Crossref PubMed Scopus (50) Google in in the of the protein that this protein not although to be with the in this may in in other be that the sialidase is not a about the of the brain for this may be that the enzyme is to the through the of the which COS-7 cells not the of the sequence suggested that are the is not this sialidase is catalytic in the is that the in the of the membrane. are the sialidase ganglioside substrates at the cell surface by its extracytoplasmic of such as and desialylation may ganglioside substrates on the of the membrane an for catalytic activity is as suggested by the on gangliosides in such as from the cell surface Glycobiology. PubMed Scopus (22) Google Scholar, K. T. Y. H. H. H. Y. K. Cell Res. 1997; PubMed Scopus Google Scholar). is that the may the sialidase an In the functional of the sialidase is not only the plasma membrane but also gangliosides are known to signal processes (19Zeller C.B. Marchase R.B. Am. J. 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