Biosynthesis and secretion of human plasma phospholipid transfer protein.
Plasma phospholipid transfer protein (PLTP) plays a critical role in lipoprotein metabolism and reverse cholesterol transport. We have studied the biosynthesis and secretion of PLTP using a stably transfected inducible HeLa cell line. Pulse-chase analysis revealed that: i) the major secreted forms of PLTP carry complex N-glycans; ii) N-glycosylation is crucial for PLTP secretion; iii) Endo H-resistant forms of PLTP could not be enriched using a 20 degrees C temperature block, indicating that the transport of PLTP from the endoplasmic reticulum to the Golgi apparatus is exceptionally sensitive to low temperatures; and iv) treatment of the PLTP-producing cells with the reducing agent dithiothreitol caused a reversible secretion arrest, suggesting a role of disulfide 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, whereas deletion of 35-50 residues results in a complete absence of secretion; and ii) the deletion of 30 C-terminal amino acid residues almost completely abolished the phospholipid transfer activity of PLTP. The present study describes for the first time the biosynthesis of phospholipid transfer protein and provides tools for detailed elucidation of the structure-function relationships in the protein.
Huuskonen
J
J
Department of Biochemistry, National Public Health Institute, Helsinki, Finland.
Jauhiainen
M
M
Ehnholm
C
C
Olkkonen
V M
VM
D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D011134:Polysaccharides / Q000032:analysis; D017510:Protein Folding; D013696:Temperature; D014162:Transfection
D002352:Carrier Proteins; D004220:Disulfides; D008565:Membrane Proteins; D010446:Peptide Fragments; D049294:Phospholipid Transfer Proteins; D011134:Polysaccharides; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase