Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Jul 1;349(Pt 1):343–351. doi: 10.1042/0264-6021:3490343

Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane.

E Monti 1, M T Bassi 1, N Papini 1, M Riboni 1, M Manzoni 1, B Venerando 1, G Croci 1, A Preti 1, A Ballabio 1, G Tettamanti 1, G Borsani 1
PMCID: PMC1221155  PMID: 10861246

Abstract

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (501.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banfi S., Guffanti A., Borsani G. How to get the best of dbEST. Trends Genet. 1998 Feb;14(2):80–81. doi: 10.1016/s0168-9525(97)01354-1. [DOI] [PubMed] [Google Scholar]
  3. Boguski M. S., Schuler G. D. ESTablishing a human transcript map. Nat Genet. 1995 Aug;10(4):369–371. doi: 10.1038/ng0895-369. [DOI] [PubMed] [Google Scholar]
  4. Bonten E., van der Spoel A., Fornerod M., Grosveld G., d'Azzo A. Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis. Genes Dev. 1996 Dec 15;10(24):3156–3169. doi: 10.1101/gad.10.24.3156. [DOI] [PubMed] [Google Scholar]
  5. Carrillo M. B., Milner C. M., Ball S. T., Snoek M., Campbell R. D. Cloning and characterization of a sialidase from the murine histocompatibility-2 complex: low levels of mRNA and a single amino acid mutation are responsible for reduced sialidase activity in mice carrying the Neu1a allele. Glycobiology. 1997 Oct;7(7):975–986. doi: 10.1093/glycob/7.7.975. [DOI] [PubMed] [Google Scholar]
  6. Chiarini A., Fiorilli A., Di Francesco L., Venerando B., Tettamanti G. Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface. Glycoconj J. 1993 Feb;10(1):64–71. doi: 10.1007/BF00731189. [DOI] [PubMed] [Google Scholar]
  7. Chiarini A., Fiorilli A., Siniscalco C., Tettamanti G., Venerando B. Solubilization of the membrane-bound sialidase from pig brain by treatment with bacterial phosphatidylinositol phospholipase C. J Neurochem. 1990 Nov;55(5):1576–1584. doi: 10.1111/j.1471-4159.1990.tb04941.x. [DOI] [PubMed] [Google Scholar]
  8. Chigorno V., Cardace G., Pitto M., Sonnino S., Ghidoni R., Tettamanti G. A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts. Anal Biochem. 1986 Mar;153(2):283–294. doi: 10.1016/0003-2697(86)90094-1. [DOI] [PubMed] [Google Scholar]
  9. Crennell S. J., Garman E. F., Laver W. G., Vimr E. R., Taylor G. L. Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9852–9856. doi: 10.1073/pnas.90.21.9852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ferrari J., Harris R., Warner T. G. Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases. Glycobiology. 1994 Jun;4(3):367–373. doi: 10.1093/glycob/4.3.367. [DOI] [PubMed] [Google Scholar]
  11. Gaskell A., Crennell S., Taylor G. The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure. 1995 Nov 15;3(11):1197–1205. doi: 10.1016/s0969-2126(01)00255-6. [DOI] [PubMed] [Google Scholar]
  12. Hata K., Wada T., Hasegawa A., Kiso M., Miyagi T. Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain. J Biochem. 1998 May;123(5):899–905. doi: 10.1093/oxfordjournals.jbchem.a022022. [DOI] [PubMed] [Google Scholar]
  13. Igdoura S. A., Gafuik C., Mertineit C., Saberi F., Pshezhetsky A. V., Potier M., Trasler J. M., Gravel R. A. Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts. Hum Mol Genet. 1998 Jan;7(1):115–121. doi: 10.1093/hmg/7.1.115. [DOI] [PubMed] [Google Scholar]
  14. Igdoura S. A., Mertineit C., Trasler J. M., Gravel R. A. Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells. Hum Mol Genet. 1999 Jun;8(6):1111–1116. doi: 10.1093/hmg/8.6.1111. [DOI] [PubMed] [Google Scholar]
  15. Kopitz J., Mühl C., Ehemann V., Lehmann C., Cantz M. Effects of cell surface ganglioside sialidase inhibition on growth control and differentiation of human neuroblastoma cells. Eur J Cell Biol. 1997 May;73(1):1–9. [PubMed] [Google Scholar]
  16. Kopitz J., von Reitzenstein C., Mühl C., Cantz M. Role of plasma membrane ganglioside sialidase of human neuroblastoma cells in growth control and differentiation. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1188–1193. doi: 10.1006/bbrc.1994.1356. [DOI] [PubMed] [Google Scholar]
  17. Lieser M., Harms E., Kern H., Bach G., Cantz M. Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties. Biochem J. 1989 May 15;260(1):69–74. doi: 10.1042/bj2600069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Milner C. M., Smith S. V., Carrillo M. B., Taylor G. L., Hollinshead M., Campbell R. D. Identification of a sialidase encoded in the human major histocompatibility complex. J Biol Chem. 1997 Feb 14;272(7):4549–4558. doi: 10.1074/jbc.272.7.4549. [DOI] [PubMed] [Google Scholar]
  20. Miyagi T., Konno K., Emori Y., Kawasaki H., Suzuki K., Yasui A., Tsuik S. Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase. J Biol Chem. 1993 Dec 15;268(35):26435–26440. [PubMed] [Google Scholar]
  21. Miyagi T., Sagawa J., Konno K., Handa S., Tsuiki S. Biochemical and immunological studies on two distinct ganglioside-hydrolyzing sialidases from the particulate fraction of rat brain. J Biochem. 1990 May;107(5):787–793. doi: 10.1093/oxfordjournals.jbchem.a123126. [DOI] [PubMed] [Google Scholar]
  22. Miyagi T., Tsuiki S. Purification and characterization of cytosolic sialidase from rat liver. J Biol Chem. 1985 Jun 10;260(11):6710–6716. [PubMed] [Google Scholar]
  23. Miyagi T., Wada T., Iwamatsu A., Hata K., Yoshikawa Y., Tokuyama S., Sawada M. Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides. J Biol Chem. 1999 Feb 19;274(8):5004–5011. doi: 10.1074/jbc.274.8.5004. [DOI] [PubMed] [Google Scholar]
  24. Monti E., Preti A., Nesti C., Ballabio A., Borsani G. Expression of a novel human sialidase encoded by the NEU2 gene. Glycobiology. 1999 Dec;9(12):1313–1321. doi: 10.1093/glycob/9.12.1313. [DOI] [PubMed] [Google Scholar]
  25. Monti E., Preti A., Rossi E., Ballabio A., Borsani G. Cloning and characterization of NEU2, a human gene homologous to rodent soluble sialidases. Genomics. 1999 Apr 1;57(1):137–143. doi: 10.1006/geno.1999.5749. [DOI] [PubMed] [Google Scholar]
  26. Ohman R., Rosenberg A., Svennerholm L. Human brain sialidase. Biochemistry. 1970 Sep 15;9(19):3774–3782. doi: 10.1021/bi00821a017. [DOI] [PubMed] [Google Scholar]
  27. Pitto M., Giglioni A., Tettamanti G. Dual subcellular localization of sialidase in cultured granule cells differentiated in culture. Neurochem Int. 1992 Oct;21(3):367–374. doi: 10.1016/0197-0186(92)90187-v. [DOI] [PubMed] [Google Scholar]
  28. Potier M., Lu Shun Yan D., Womack J. E. Neuraminidase deficiency in the mouse. FEBS Lett. 1979 Dec 15;108(2):345–348. doi: 10.1016/0014-5793(79)80560-8. [DOI] [PubMed] [Google Scholar]
  29. Pshezhetsky A. V., Richard C., Michaud L., Igdoura S., Wang S., Elsliger M. A., Qu J., Leclerc D., Gravel R., Dallaire L. Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis. Nat Genet. 1997 Mar;15(3):316–320. doi: 10.1038/ng0397-316. [DOI] [PubMed] [Google Scholar]
  30. Riboni L., Prinetti A., Bassi R., Tettamanti G. Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane. FEBS Lett. 1991 Aug 5;287(1-2):42–46. doi: 10.1016/0014-5793(91)80012-r. [DOI] [PubMed] [Google Scholar]
  31. Rost B., Casadio R., Fariselli P., Sander C. Transmembrane helices predicted at 95% accuracy. Protein Sci. 1995 Mar;4(3):521–533. doi: 10.1002/pro.5560040318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rottier R. J., Bonten E., d'Azzo A. A point mutation in the neu-1 locus causes the neuraminidase defect in the SM/J mouse. Hum Mol Genet. 1998 Feb;7(2):313–321. doi: 10.1093/hmg/7.2.313. [DOI] [PubMed] [Google Scholar]
  33. Saito M., Yu R. K. Further characterization of a myelin-associated neuraminidase: properties and substrate specificity. J Neurochem. 1986 Aug;47(2):632–641. doi: 10.1111/j.1471-4159.1986.tb04547.x. [DOI] [PubMed] [Google Scholar]
  34. Sonnino S., Ghidoni R., Gazzotti G., Kirschner G., Galli G., Tettamanti G. High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition. J Lipid Res. 1984 Jun;25(6):620–629. [PubMed] [Google Scholar]
  35. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Taylor G. Sialidases: structures, biological significance and therapeutic potential. Curr Opin Struct Biol. 1996 Dec;6(6):830–837. doi: 10.1016/s0959-440x(96)80014-5. [DOI] [PubMed] [Google Scholar]
  37. Tettamanti G., Bonali F., Marchesini S., Zambotti V. A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim Biophys Acta. 1973 Jan 19;296(1):160–170. doi: 10.1016/0005-2760(73)90055-6. [DOI] [PubMed] [Google Scholar]
  38. Tettamanti G., Riboni L. Gangliosides and modulation of the function of neural cells. Adv Lipid Res. 1993;25:235–267. [PubMed] [Google Scholar]
  39. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Venerando B., Fiorilli A., Croci G. L., Tettamanti G. Presence in human erythrocyte membranes of a novel form of sialidase acting optimally at neutral pH. Blood. 1997 Sep 1;90(5):2047–2056. [PubMed] [Google Scholar]
  41. Wada T., Yoshikawa Y., Tokuyama S., Kuwabara M., Akita H., Miyagi T. Cloning, expression, and chromosomal mapping of a human ganglioside sialidase. Biochem Biophys Res Commun. 1999 Jul 22;261(1):21–27. doi: 10.1006/bbrc.1999.0973. [DOI] [PubMed] [Google Scholar]
  42. Warner T. G., Chang J., Ferrari J., Harris R., McNerney T., Bennett G., Burnier J., Sliwkowski M. B. Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells. Glycobiology. 1993 Oct;3(5):455–463. doi: 10.1093/glycob/3.5.455. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES