Microsomal Triglyceride Transfer Protein Binding and Lipid Transfer Activities Are Independent of Each Other, but Both Are Required for Secretion of Apolipoprotein B Lipoproteins from Liver Cells

Recent studies indicate that microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) interact physically via two specific binding sites located within the amino-terminal globular region of apoB100. The first site is thought to be within the first 5.8% of the amino-terminal sequence, and the second site is between 9 and 16% of the amino-terminal sequence. It is not clear from prior studies whether these sites have unique or overlapping functions. Furthermore, there are no data differentiating between lipid transfer and potential chaperone functions of MTP. In the present study we have attempted to further characterize the physiologic interaction between apoB and MTP and to determine the relationship between the binding and lipid transfer aspects of the interaction. HepG2 cells were transiently transfected with apoB cDNAs, and MTP binding to apoB polypeptides was determined by two-step immunoprecipitation. MTP bound equally well to apoB polypeptides with (apoB13, 16,beta, apoB34, and apoB42) or without (apoB16, apoB13, and 16 or apoB13, 13, and 16) beta sheet domains. When proteasomal degradation of newly synthesized apoB polypeptides was blocked, MTP binding to all of the apoB polypeptides was only modestly affected by lipid availability and was independent of MTP-associated lipid transfer. Furthermore, MTP did not bind directly to a portion of the first beta sheet domain. We created two apoB constructs (apoB16del and apoB34del) by deleting the first 210 amino acids of apoB16 and apoB34. These apoB polypeptides, therefore, lacked the putative first MTP binding site. MTP binding to apoB16del and apoB34del was decreased significantly. However, the secretion of apoB16del was not different from apoB16, whereas the secretion of apoB34del was impaired significantly. Our results indicate that the interaction between MTP and apoB involves independent binding and lipid transfer activities but that both activities are required for the secretion of apolipoprotein B from liver cells.