HUMAN PLASMA CHOLESTERYL ESTER TRANSFER PROTEIN CONSISTS OF A MIXTUREOF 2 FORMS REFLECTING VARIABLE GLYCOSYLATION AT ASPARAGINE-341

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids and phospholipids between the plasma lipoproteins. The deduced M(r) of the CETP polypeptide from the CDNA is 53 000, but in sodium dodecyl sulfate (SDS) gels plasma CETP appears as a broad ba nd containing two different moelcular forms of M(r) 65 000-71 000. The purpose of this study was to see if variable N-linked glycosylation c ould explain the microheterogeneity of CETP. Recombinant CETP (rCETP), derived from stable expression of the CETP cDNA in Chinese hamster ov ary (CHO) cells, appeared as a protein doublet comparable to plasma CE TP. Digestion of plasma or rCETP with N-glycosidase F (glyco F, to rem ove N-linked carbohydrates) resulted in the formation of a lower M(r) doublet in which the bottom band approximated the M(r) of the CETP pol ypeptide. Metabolic labeling of the rCETP with [H-3]mannose and [H-3]g lucosamine, followed by digestion with glyco F, suggested that the top band of the doublet contained residual N-linked carbohydrates resista nt to glyco F digestion. To explore this hypothesis further, each of t he four potential N-linked glycosylation sites of CETP (at amino acid positions 88, 240, 341, and 396) was eliminated by mutagenesis of aspa ragine to glutamine. The wild-type (WT) and mutant CETP cDNAs were tra nsiently expressed in COS-7 cells. Each mutant CETP showed a lower M(r ) than WT, indicating that all four sites were occupied by N-linked ca rbohydrate. Each mutant and WT protein appeared as a doublet except fo r the 341N --> Q mutant, which gave rise to a single protein band coin cident with the lower band of the doublet of WT CETP. Furthermore, dig estion of the 341N --> Q protein with glyco F gave rise to a single ba nd of M(r) approximately 53 000. The 88N --> Q and 396N --> Q mutants were poorly secreted, but the 341N --> Q protein was well secreted and displayed moderately increased cholesteryl ester transfer activity co mpared to WT. The results suggest that plasma or rCETP consists of a m ixture of two forms in which amino acid 341 is or is not occupied by N -linked carbohydrate. Although a preliminary survey indicated only sli ght variations in normolipidemic subjects, variable ratios of the two forms in altered metabolic states could give rise to differences in th e specific activity of plasma CETP.