Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B-100-containing lipoproteins.
Apolipoprotein B-100 (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL). The mechanism of VLDL assembly was explored by perturbing apoB folding in HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Although apoB contains eight known disulfide bonds, seven of which are positioned in the amino-terminal 21% of the protein, its assembly and secretion was only partially blocked in cells treated with 2 mM DTT, a condition that fully blocks the secretion of other disulfide-bonded proteins. Nonreducing gel electrophoresis of an apoB-derived proteolytic peptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation to a DTT-resistant form after completing synthesis of only the first approximately 20-25% of the protein. If, however, DTT was used under conditions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked. Reduced forms of apoB were extremely labile and, unlike other disulfide-bonded proteins, incapable of achieving secretion competence posttranslationally. These results indicate that disulfide bond formation within the amino-terminus of apoB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascent polypeptide chain is consistent with a model in which the amino-terminal domain of apoB undergoes an independent folding and maturation process, the completion of which may represent an initiation phase of triglyceride-rich lipoprotein assembly.
Shelness
G S
GS
Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27157-1040, USA.
Thornburg
J T
JT
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000096:biosynthesis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D013917:Thrombin / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D004220:Disulfides; D008079:Lipoproteins, VLDL; D013917:Thrombin; D004229:Dithiothreitol