Evidence for a structural relationship between apoB75kDa and human plasma apolipoprotein B 100, from translation of human liver mRNA in vitro and immunochemical studies with monoclonal and polyclonal antibodies

Abstract

We have investigated the relation between an 80-kDa protein synthesized in vitro in protein-synthesizing system programmed with human liver mRNA [Olofsson, S.-O., Elias, P., Boström, K., Lundholm, K., Norfeldt, P.-I., Wiklund, O., Fager, G., and Bondjers, G. (1983) FEBS Lett. 156, 63-66] and a 70-80-kDa protein, apoB75kDa, isolated from the low-density lipoproteins-2 (LDL-2) [Olofson, S.-O., Boström, K., Svanberg, U., and Bondjers, G. (1980) Biochemistry 19, 1059-1064]. 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]-methionine) with Staphylococcus aureus V8 protease generated six [35S]-methionine-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 two proteins. Taken together, our 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). We have also compared apoB75kDa and the major component of apoLDL-2, apoB100 [Kane, J. P., Hardman, D.A., and Paulus, H.E. (1980) Proc. Natl Acad. Sci USA 77, 2465-2469] by immunochemical methods. We could demonstrate that six monoclonal antibodies directed against four to six 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 two proteins. Taken together our results support the hypothesis that apoB100 has a subunit structure. We therefore suggest that apoB75kDa is a subunit of apoB100 synthesized in human liver.