The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion.

UNLABELLED: Secretion of apolipoprotein B (apoB) from mammalian cells requires the presence of functional microsomal triglyceride transfer protein (MTP). We previously reported that co-expressing the human intestinal form of apoB, B48, with both subunits of human MTP in oleate-treated Sf21 cells led to a dramatic induction of B48 secretion. Deletion mutagenesis studies showed that the cysteine-enriched amino terminus of apoB was necessary for the MTP responsiveness (Gretch, D. G., Sturley, S. L., Wang, L., Dunning, A., Grunwald, K. A. A., Wetterau, J. R., Yao, Z., Talmud, P., and Attie, A. D. (1996) J. Biol. Chem. 271, 8682-8691). We therefore hypothesized that the small subunit of MTP, protein-disulfide isomerase (PDI), plays a role in apoB secretion by facilitating correct disulfide bond formation. To determine whether the enzymatic activities of PDI are important for MTP-stimulated apoB secretion, the wild type PDI subunit was replaced with an active site mutant, mPDI (Cys36 --> Ser/Cys380 --> Ser), lacking both disulfide shuffling and redox activities. MTP containing mPDI was fully functional in promoting apoB and triglyceride secretion. Therefore, the shufflase and redox activities of PDI are not necessary for the function of MTP. Since PDI exists in large molar excess over the other subunit of MTP, the role of free PDI (independent of the MTP complex) was investigated. PDI or mPDI was co-expressed with B48 and B17, a fragment encompassing the amino-terminal 17% of apoB. Mutant PDI significantly and specifically reduced the accumulation of the B17 and B48 both intracellularly and in the culture medium. The reduction was partially eliminated by the protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, consistent with rapid co- or post-translational degradation of apoB in the presence of mPDI. Treating the cells with oleate reversed the effect of mPDI on B48 secretion in a dose-dependent manner, but had no effect on B17. IN
CONCLUSION: 1) the role of PDI in the MTP complex involves functions other than its known enzymatic activities; 2) one or both of the enzymatic activities of free PDI is/are important for the MTP-independent steps of apoB secretion; 3) oleate can affect apoB secretion at high physiological concentrations and compensate for the insufficiency of PDI activities.