Evidence for a structural relationship between apoB75 kDa and human plasma apolipoprotein B100, from translation of human liver mRNA in vitro and immunochemical studies with monoclonal and polyclonal antibodies

Abstract

The relation between an 80-kDa [kilodalton] protein synthesized in vitro in protein-synthesizing system programmed with human liver mRNA and a 70-80-kDa protein, apoB75kDa, from the low-density lipoproteins-2 (LDL-2) was investigated. Five monoclonal antibodies directed against LDL-2 as well as polyclonal antibodies against a narrow density cut of LDL-2 (d = 1.030-1.055) were used to precipitate apoB-related proteins synthesized in vitro in a protein-synthesizing system programmed with human liver mRNA (or total RNA fraction). With all monoclonal antibodies as well as the polyclonal antibodies, a protein with an estimated molecular mass of 80 .+-. 1.3 kDa (mean .+-. SD, n = 12) could be precipitated. The observation that all monoclonal antibodies used reacted with apoB75kDa indicates a close immunological relation between this 80-kDa protein and apoB75kDa. Limited proteolysis of the 80-kDa protein (synthesized in the presence of [35S]-Met) with Staphylococcus aureus V8 protease generated 6 [35S]-Met-containing bands that could be separated on a polyacrylamide gradient gel (12-20%). All these radioactive bands corresponded to major protein-stained bands obtained after limited proteolysis of apoB75kDa. This observation suggests a structural relation between the 2 proteins. Taken together, the results indicate that a protein corresponding to apoB75kDa is synthesized in vitro in a protein synthesizing system programmed with human liver mRNA (or total RNA fraction). The apoB75kDa and the major component of apoLDL-2, apoB100 were compared by immunochemical methods. Six monoclonal antibodies directed against 4-6 different epitopes on LDL-2, as well as polyclonal antibodies to apoB100 and apoB75kDa, all reacted with apoB75kDa and apoB100. These observations indicate a close immunological relation between the 2 protein. Taken together the results support the hypothesis that apoB100 has a subunit structure. The apoB75kDa may be a subunit of apoB100 synthesized in human liver.