{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T04:24:18Z","timestamp":1781756658002,"version":"3.54.5"},"reference-count":44,"publisher":"Portland Press Ltd.","issue":"1","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2005,8,15]]},"abstract":"<jats:p>The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370\u2192Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (\u223c20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.<\/jats:p>","DOI":"10.1042\/bj20050325","type":"journal-article","created":{"date-parts":[[2005,8,10]],"date-time":"2005-08-10T14:29:33Z","timestamp":1123684173000},"page":"95-103","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":41,"title":["The N370S (Asn370\u2192Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C"],"prefix":"10.1042","volume":"390","author":[{"given":"Rosa","family":"Salvioli","sequence":"first","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Massimo","family":"Tatti","sequence":"additional","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanna","family":"Scarpa","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Pathology, University of Rome \u2018La Sapienza\u2019, Viale Regina Elena 234, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabrina\u00a0Maria","family":"Moavero","sequence":"additional","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fiorella","family":"Ciaffoni","sequence":"additional","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federica","family":"Felicetti","sequence":"additional","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine\u00a0R.","family":"Kaneski","sequence":"additional","affiliation":[{"name":"Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roscoe\u00a0O.","family":"Brady","sequence":"additional","affiliation":[{"name":"Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna\u00a0Maria","family":"Vaccaro","sequence":"additional","affiliation":[{"name":"Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita', Viale Regina Elena 299, 00161 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2005,8,9]]},"reference":[{"key":"2021112213244857100_B1","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/0006-291X(65)90743-6","article-title":"Metabolism of glucocerebrosides. II. Evidence of an enzymatic deficiency in Gaucher's disease","volume":"18","author":"Brady","year":"1965","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2021112213244857100_B2","doi-asserted-by":"crossref","first-page":"1048","DOI":"10.1016\/0006-291X(76)90760-9","article-title":"Solubilization of glucocerebrosidase from human placenta and demonstration of a phospholipid requirement for its catalytic activity","volume":"71","author":"Dale","year":"1976","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2021112213244857100_B3","first-page":"5","article-title":"Mammalian glucocerebrosidase: implication for Gaucher disease","volume":"58","author":"Glew","year":"1988","journal-title":"Lab. Invest."},{"key":"2021112213244857100_B4","doi-asserted-by":"crossref","first-page":"2810","DOI":"10.1073\/pnas.68.11.2810","article-title":"Gaucher's disease: deficiency of \u2018acid\u2019-glucosidase and reconstitution of activity in vitro","volume":"68","author":"Ho","year":"1971","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021112213244857100_B5","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1042\/bj1360821","article-title":"Glucocerebrosidase: reconstitution from macromolecular components depends on acidic phospholipids","volume":"136","author":"Ho","year":"1973","journal-title":"Biochem. J."},{"key":"2021112213244857100_B6","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1096\/fasebj.5.3.2001789","article-title":"Saposin proteins: structure, function, and role in human lysosomal storage disorders","volume":"5","author":"O'Brien","year":"1991","journal-title":"FASEB J."},{"key":"2021112213244857100_B7","doi-asserted-by":"crossref","first-page":"999","DOI":"10.1007\/BF01716064","article-title":"Activator protein deficient Gaucher disease","volume":"67","author":"Christomanou","year":"1989","journal-title":"Klin. Wochenschr."},{"key":"2021112213244857100_B8","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/0014-5793(91)80760-Z","article-title":"Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease","volume":"284","author":"Schnabel","year":"1991","journal-title":"FEBS Lett."},{"key":"2021112213244857100_B9","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/0014-5793(93)81631-9","article-title":"Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes","volume":"336","author":"Vaccaro","year":"1993","journal-title":"FEBS Lett."},{"key":"2021112213244857100_B10","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/S0014-5793(00)01417-4","article-title":"Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids","volume":"472","author":"Salvioli","year":"2000","journal-title":"FEBS Lett."},{"key":"2021112213244857100_B11","doi-asserted-by":"crossref","first-page":"30576","DOI":"10.1074\/jbc.270.51.30576","article-title":"pH-dependent conformational properties of saposins and their interactions with phospholipid membranes","volume":"270","author":"Vaccaro","year":"1995","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B12","doi-asserted-by":"crossref","first-page":"14729","DOI":"10.1021\/bi0301338","article-title":"Solution structure of human saposin C: pH-dependent interaction with phospholipid vesicles","volume":"42","author":"de\u00a0Alba","year":"2003","journal-title":"Biochemistry"},{"key":"2021112213244857100_B13","doi-asserted-by":"crossref","first-page":"16862","DOI":"10.1074\/jbc.272.27.16862","article-title":"Effect of saposins A and C on the enzymatic hydrolysis of liposomal glucosylceramide","volume":"272","author":"Vaccaro","year":"1997","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B14","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1002\/(SICI)1098-1004(1996)8:3<207::AID-HUMU2>3.0.CO;2-6","article-title":"Glucocerebrosidase (Gaucher disease)","volume":"8","author":"Beutler","year":"1996","journal-title":"Hum. Mutat."},{"key":"2021112213244857100_B15","first-page":"3635","article-title":"Gaucher disease","volume-title":"The Metabolic and Molecular Bases of Inherited Disease","author":"Beutler","year":"2001"},{"key":"2021112213244857100_B16","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1016\/S0950-3536(97)80032-7","article-title":"Gaucher's disease: molecular, genetic and enzymological aspects","volume":"10","author":"Grabowski","year":"1997","journal-title":"Baillieres Clin. Haematol."},{"key":"2021112213244857100_B17","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/0167-4838(90)90122-V","article-title":"Conditions affecting the activity of glucocerebrosidase purified from spleens of control subjects and patients with type 1 Gaucher disease","volume":"1041","author":"Aerts","year":"1990","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213244857100_B18","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1172\/JCI116276","article-title":"Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease","volume":"91","author":"van\u00a0Weely","year":"1993","journal-title":"J. Clin. Invest."},{"key":"2021112213244857100_B19","doi-asserted-by":"crossref","first-page":"3661","DOI":"10.1016\/S0021-9258(19)67845-2","article-title":"Characterization of human glucocerebrosidase from different mutant alleles","volume":"266","author":"Ohashi","year":"1991","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B20","doi-asserted-by":"crossref","first-page":"6827","DOI":"10.1016\/S0021-9258(19)39223-3","article-title":"Analyses of catalytic activity and inhibitor binding of human acid \u03b2-glucosidase by site-directed mutagenesis","volume":"265","author":"Grace","year":"1990","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B21","doi-asserted-by":"crossref","first-page":"2283","DOI":"10.1016\/S0021-9258(17)42166-1","article-title":"Analysis of human acid \u03b2-glucosidase by site-directed mutagenesis and heterologous expression","volume":"269","author":"Grace","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B22","doi-asserted-by":"crossref","first-page":"17674","DOI":"10.1074\/jbc.M313517200","article-title":"Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C","volume":"279","author":"Salvioli","year":"2004","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B23","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/0003-2697(86)90043-6","article-title":"A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibody","volume":"154","author":"Aerts","year":"1986","journal-title":"Anal. Biochem."},{"key":"2021112213244857100_B24","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature (London)"},{"key":"2021112213244857100_B25","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/0925-4439(91)90066-I","article-title":"Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts","volume":"1096","author":"van\u00a0Weely","year":"1991","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213244857100_B26","doi-asserted-by":"crossref","first-page":"14319","DOI":"10.1016\/S0021-9258(17)38720-3","article-title":"Biosynthesis of the lysosomal enzyme glucocerebrosidase","volume":"260","author":"Erickson","year":"1985","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B27","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1111\/j.1432-1033.1987.tb11008.x","article-title":"Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts","volume":"164","author":"Jonsson","year":"1987","journal-title":"Eur. J. Biochem."},{"key":"2021112213244857100_B28","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1111\/j.1432-1033.1990.tb19173.x","article-title":"Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase","volume":"191","author":"van\u00a0Weely","year":"1990","journal-title":"Eur. J. Biochem."},{"key":"2021112213244857100_B29","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1038\/sj.embor.embor873","article-title":"X-ray structure of human acid-\u03b2-glucosidase, the defective enzyme in Gaucher disease","volume":"4","author":"Dvir","year":"2003","journal-title":"EMBO Rep."},{"key":"2021112213244857100_B30","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1038\/32440","article-title":"A lipid associated with the antiphospholipid syndrome regulates endosome structure and function","volume":"392","author":"Kobayashi","year":"1998","journal-title":"Nature (London)"},{"key":"2021112213244857100_B31","doi-asserted-by":"crossref","first-page":"32157","DOI":"10.1074\/jbc.M202838200","article-title":"Separation and characterization of late endosomal membrane domains","volume":"277","author":"Kobayashi","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B32","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1007\/BF00314009","article-title":"Ultrastuctural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry","volume":"234","author":"Willemsen","year":"1987","journal-title":"J. Neurol."},{"key":"2021112213244857100_B33","first-page":"157","article-title":"Immunocytochemical localization of sphingolipid activator protein 2 (Sap 2) in normal and Sap-deficient fibroblasts","volume":"51","author":"Paton","year":"1990","journal-title":"Eur. J. Cell Biol."},{"key":"2021112213244857100_B34","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1038\/14084","article-title":"Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases","volume":"1","author":"Puri","year":"1999","journal-title":"Nat. Cell Biol."},{"key":"2021112213244857100_B35","doi-asserted-by":"crossref","first-page":"20961","DOI":"10.1074\/jbc.M300304200","article-title":"Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage","volume":"278","author":"Puri","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021112213244857100_B36","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1038\/35096054","article-title":"The endocytic pathway: a mosaic of domains","volume":"2","author":"Gruenberg","year":"2001","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2021112213244857100_B37","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1006\/scdb.1998.0257","article-title":"Lipids, lipid domains and lipid-protein interactions in endocytic membrane traffic","volume":"9","author":"Kobayashi","year":"1998","journal-title":"Semin. Cell Dev. Biol."},{"key":"2021112213244857100_B38","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1038\/10084","article-title":"Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport","volume":"1","author":"Kobayashi","year":"1999","journal-title":"Nat. Cell Biol."},{"key":"2021112213244857100_B39","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1023\/A:1022530508763","article-title":"Saposins and their interaction with lipids","volume":"24","author":"Vaccaro","year":"1999","journal-title":"Neurochem. Res."},{"key":"2021112213244857100_B40","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/0005-2760(83)90218-7","article-title":"Biochemical studies in Niemann-Pick disease I. Major sphingolipids of liver and spleen","volume":"750","author":"Vanier","year":"1983","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213244857100_B41","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1023\/A:1022575511354","article-title":"Lipid changes in Niemann-Pick C disease type C brain: personal experience and review of the literature","volume":"24","author":"Vanier","year":"1999","journal-title":"Neurochem. Res."},{"key":"2021112213244857100_B42","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1146\/annurev.bi.55.070186.003311","article-title":"Acidification of the endocytic and exocytic pathways","volume":"55","author":"Mellman","year":"1986","journal-title":"Annu. Rev. Biochem."},{"key":"2021112213244857100_B43","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1242\/jeb.172.1.39","article-title":"The importance of being acid: the role of acidification in intracellular membrane traffic","volume":"172","author":"Mellman","year":"1992","journal-title":"J. Exp. Biol."},{"key":"2021112213244857100_B44","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1146\/annurev.cb.05.110189.002411","article-title":"The biogenesis of lysosomes","volume":"5","author":"Kornfeld","year":"1989","journal-title":"Annu. Rev. Cell Biol."}],"container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/390\/1\/95\/797378\/bj3900095.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/390\/1\/95\/797378\/bj3900095.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,22]],"date-time":"2021-11-22T14:03:56Z","timestamp":1637589836000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/390\/1\/95\/42042\/The-N370S-Asn370-Ser-mutation-affects-the-capacity"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2005,8,9]]},"references-count":44,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2005,8,15]]}},"URL":"https:\/\/doi.org\/10.1042\/bj20050325","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[2005,8,9]]}}}