Human plasma cholesteryl ester transfer protein consists of a mixture of two 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 band containing two different molecular forms of M(r) 65,000-71,000. The purpose of this study was to see if variable N-linked glycosylation could explain the microheterogeneity of CETP. Recombinant CETP (rCETP), derived from stable expression of the CETP cDNA in Chinese hamster ovary (CHO) cells, appeared as a protein doublet comparable to plasma CETP. Digestion of plasma or rCETP with N-glycosidase F (glyco F, to remove 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 polypeptide. Metabolic labeling of the rCETP with [3H]mannose and [3H]glucosamine, followed by digestion with glyco F, suggested that the top band of the doublet contained residual N-linked carbohydrates resistant to glyco F digestion. To explore this hypothesis further, each of the four potential N-linked glycosylation sites of CETP (at amino acid positions 88, 240, 341, and 396) was eliminated by mutagenesis of asparagine to glutamine. The wild-type (WT) and mutant CETP cDNAs were transiently 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 carbohydrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
S C
SC
Stevenson
S
S
Wang
L
L
Deng
A R
AR
Tall
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D053480:Cholesterol Ester Transfer Proteins; D006224:Cricetinae; D005260:Female; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D001216:Asparagine; D006026:Glycoside Hydrolases; D009439:Neuraminidase