title: N-Glycosylation is required for secretion-competent human plasma phospholipid transfer protein.
abstract: Human plasma phospholipid transfer protein (PLTP) contains six potential N-glycosylation sites (Asn-X-Ser). To study the role of these sites on PLTP structure and function, seven variants in which asparagine (N) residues were converted to glycine (G) were prepared by site-directed mutagenesis. These were N(47)G, N(77)G, N(100)G, N(126)G, N(228)G, N(381)G and N(47, 77, 100, 126, 228, 381)G (N(null)G). These variants and wild-type (WT) PLTP were expressed in COS-7 cells. Intracellular and secreted PLTP mass was analyzed by Western blots and quantitative enzyme-linked immunosorbent assay; PLTP activities in cellular lysates and media were based on the transfer of [(3)H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N(null)G was not detected intracellularly. N(381)G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N(47)G, N(77)G, N(100)G, N(126)G, N(228)G and N(381)G were similar in cell lysate (range = 67-90% WT) and medium (range = 65-77% WT). Intracellular masses of these PLTP variants were similar to that of WT (Mean = 103% WT); mean secreted mass was 88% WT. These results suggest that secretion-competent PLTP requires glycosylation but that no single glycosylation site is required.
authors.affiliation: MS A-601, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Shi-Jing
authors.initials: SJ
authors.lastname: Qu
authors.forename: Hui-Zhen
authors.initials: HZ
authors.lastname: Fan
authors.forename: Baiba K
authors.initials: BK
authors.lastname: Gillard
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Pownall
mesh_terms: D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D049294:Phospholipid Transfer Proteins / Q000097:blood* / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation
chemical_list: C493430:PLTP protein, human; D049294:Phospholipid Transfer Proteins