FUNCTIONAL-ANALYSIS OF DISULFIDE LINKAGES CLUSTERED WITHIN THE AMINO-TERMINUS OF HUMAN APOLIPOPROTEIN-B

We tested the involvement of N-terminal six disulfide bonds (Cys-1 thr ough Cys-12) of human apolipoprotein (apo) B in the assembly and secre tion of lipoproteins using two C-terminal truncated apoB variants, nam ely B50 and B18, In transfected rat hepatoma McA-RH7777 cells, B50 cou ld assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins, When all 12 cysteine residues were subs tituted with alanines in B50, the mutant protein (B50C1-12) lost its a bility to assemble lipid and was degraded intracellularly, However, mu tation had no effect on B50C1-12 translation or translocation across t he microsomal membrane, Post-translational degradation of B50C1-12 was partially inhibited by the proteasome inhibitor MG132. To determine w hich cysteines were critical in VLDL assembly and secretion, we prepar ed 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 o f disulfide bond formation within Cys-8 to Cys-8 diminished apoB secre tion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or n o 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 o f VLDL containing endogenous B100 or B48 were impaired in cells transf ected with B50s containing Cys-7 and Cys-8 mutation, The Cys-to-Ala su bstitution abolished recognition of B50 by MB19, a conformational anti body with an epitope at the N terminus of human apoB, The Cys-to-Ala s ubstitution also attenuated secretion of B18, but the effect of the mu tation on B18 secretion was less evident than on B50.