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). Th e mechanism of VLDL assembly was explored by perturbing apoB folding i n HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Alth ough apoB contains eight known disulfide bonds, seven of which are pos itioned in the amino-terminal 21% of the protein, its assembly and sec retion was only partially blocked in cells treated with 2 mM DTT, a co ndition that fully blocks the secretion of other disulfide-bonded prot eins. Nonreducing gel electrophoresis of an apoB-derived proteolytic p eptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation t o a DTT-resistant form after completing synthesis of only the first si milar to 20-25% of the protein. If, however, DTT was used under condit ions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked, Reduced forms of apoB were e xtremely 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 ap oB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascen t polypeptide chain is consistent with a model in which the amino-term inal domain of apoB undergoes an independent folding and maturation pr ocess, the completion of which may represent an initiation phase of tr iglyceride-rich lipoprotein assembly.