Human plasma cholesteryl ester transfer protein consists of a mixture of two forms reflecting variable glycosylation at asparagine 341

• Published in: Biochemistry (Easton) Vol. 32; no. 19; pp. 5121 - 5126
• Main Authors: Stevenson, Susan C, Wang, Suke, Deng, Liping, Tall, Alan R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18.05.1993
• Subjects: Analytical, structural and metabolic biochemistry; Animals; ASPARAGINA; ASPARAGINE; Asparagine - metabolism; Base Sequence; Binding and carrier proteins; binding proteins; biochemical polymorphism; Biological and medical sciences; blood; blood proteins; Carrier Proteins; Carrier Proteins - blood; Carrier Proteins - chemistry; Carrier Proteins - genetics; chemical reactions; chemistry; CHO Cells; CHOLESTEROL; Cholesterol Ester Transfer Proteins; cholesteryl esters; COLESTEROL; Cricetinae; ESTER; ESTERES; Female; Fundamental and applied biological sciences. Psychology; GENERO HUMANO; genetics; GENRE HUMAIN; Glycoproteins; Glycoside Hydrolases; Glycoside Hydrolases - metabolism; Glycosylation; Humans; Male; metabolism; Molecular Sequence Data; Molecular Weight; MUTACION; Mutagenesis, Site-Directed; MUTATION; Neuraminidase; Neuraminidase - metabolism; POLIMORFISMO BIOQUIMICO; POLYMORPHISME BIOCHIMIQUE; PROTEINAS AGLUTINANTES; PROTEINAS SANGUINEAS; PROTEINE DE LIAISON; PROTEINE SANGUINE; Proteins; REACCIONES QUIMICAS; REACTION CHIMIQUE; Recombinant Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; site-directed mutagenesis; Human; Molecular form; Glycosylation; Molecular structure; Carrier protein
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00070a021

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids and phospholipids between the plasma lipoproteins. The deduced Mr 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 Mr 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 Mr doublet in which the bottom band approximated the Mr 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 Mr than WT, indicating that all four sites were occupied by N-linked carbohydrate. Each mutant and WT protein appeared as a doublet except for the 341 N leads to Q mutant, which gave rise to a single protein band coincident with the lower band of the doublet of WT CETP. Furthermore, digestion of the 341N leads to Q protein with glyco F gave rise to a single band of Mr approximately 53 000. The 88N leads to Q and 396N leads to Q mutants were poorly secreted, but the 341N leads to Q protein was well secreted and displayed moderately increased cholesteryl ester transfer activity compared to WT