BIOSYNTHESIS AND SECRETION OF HUMAN PLASMA PHOSPHOLIPID TRANSFER PROTEIN

Plasma phospholipid transfer protein (PLTP) plays a critical role in l ipoprotein metabolism and reverse cholesterol transport, We have studi ed the biosynthesis and secretion of PLTP using a stably transfected i nducible HeLa cell line. Pulse-chase analysis revealed that: i) the ma jor secreted forms of PLTP carry complex N-glycans; ii) N-glycosylatio n is crucial for PLTP! secretion; iii) Endo H-resistant forms of PLTP could not be enriched using a 20 degrees C temperature block, indicati ng that the transport of PLTP from the endoplasmic reticulum to-the Go lgi apparatus is exceptionally sensitive to low temperatures; and iv) treatment of the PLTP-producing cells with the reducing agent dithioth reitol caused a reversible secretion arrest, suggesting a role of disu lfide bonds in the correct folding of PLTP. Transient expression of C- terminally truncated PLTP variants in COS cells demonstrated that: i) the 30 C-terminal amino acids are dispensable for PLTP secretion, wher eas deletion of 35-50 residues results in a complete absence of secret ion; and ii) the deletion of 30 C-terminal amino acid residues almost completely abolished the phospholipid transfer activity of PLTP. The p resent study describes for the first time the biosynthesis of phosphol ipid transfer protein and provides tools for detailed elucidation of t he structure-function relationships in the protein.