Abstract
We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1-12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1-12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1-12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50.
Highlights
Despite the absence of information concerning specific sequence elements involved in very low density lipoproteins (VLDL) assembly, several experimental observations suggest that the N-terminal 17% of apoB100 is critical in lipoprotein formation
It has been postulated that folding of the N-terminal region, presumably mediated through disulfide bonding, is essential density lipoproteins; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; PAGE, polyacrylamide gel electrophoresis; RARE, RecA-assisted restriction endonuclease; kb, kilobase(s)
The present results suggest that an interaction between the disulfide-bonded domain and the downstream lipid binding sequences may play an important role in the post-translational stability of apoB and in VLDL assembly and secretion
Summary
Materials—Restriction and DNA modification enzymes and endoglycosidase H were purchased from New England Biolabs. To create pZeroC1–12 in which all 12 cysteines (i.e. Cys-1 to Cys-12) were changed to alanines, a fragment encompassing Cys-1 to Cys-4 mutations was excised from pZeroC1– 4 by digestion with EcoRI (in the polylinker of pCMV5) and Bsu36I (nucleotide 449 of the apoB cDNA) and ligated to pZeroC5– 8 that had been digested with the same enzymes to generate pZeroC1– 8. A XmnI-HindIII fragment (nucleotides 1547–2279 of the apoB cDNA) containing Cys-9 to Cys-12 mutations was excised from pZeroC9 –12 and used to replace the identical sequence in pZeroC1– 8 to generate the resulting pZeroC1–12. The mutated 2.45-kb EcoRI-HindIII fragments containing various Cys-to-Ala substitutions were inserted into pB50L-L after RARE cleavage with oligomers RARE-1 and RARE-2 to create apoB50 or into pCMV5 to create apoB18 expression plasmids.
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