N-Glycosylation is Required for Secretion-Competent Human Plasma Phospholipid Transfer Protein

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 N₄₇G, N₇₇G, N₁₀₀G, N₁₂₆G, N₂₂₈G, N₃₈₁G and N_{47, 77, 100, 126, 228, 381}G (N_nullG). 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 ³H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N_nullG was not detected intracellularly. N₃₈₁G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N₄₇G, N₇₇G, N₁₀₀G, N₁₂₆G, N₂₂₈G and N₃₈₁G 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.