Role of cysteine residues in human plasma phospholipid transfer protein.
Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys135 and Cys175; these residues, which correspond to Cys129 and Cys168 in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV.SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys129 --> Gly and PLTP Cys168 --> Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys5 --> Gly and PLTP Cys318 --> Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys5 --> Gly and PLTP Cys318 --> Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.
Qu
S J
SJ
Department of Medicine, Baylor College of Medicine, and The Methodist Hospital, Houston, Texas 77030, USA.
Fan
H Z
HZ
Kilinc
C
C
Pownall
H J
HJ
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D049294:Phospholipid Transfer Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D003545:Cysteine