title: Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme.
abstract: To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.
authors: Department of Ophthalmology, School of Medicine, Sapporo Medical University, Sapporo, Japan.
authors: Miki
authors: M
authors: Hiraoka
authors: Ken
authors: K
authors: Okamoto
authors: Hiroshi
authors: H
authors: Ohguro
authors: Akira
authors: A
authors: Abe
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D054467:Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D014415:Tunicamycin; D054467:Phospholipases A2; D009584:Nitrogen
keywords: N-glycosylation; lysosomal phospholipase A2; site-direct mutagenesis
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