Functional Analysis of Disulfide Linkages Clustered within the Amino Terminus of Human Apolipoprotein B
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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.
