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

We have investigated the relation between an XO-kDa protein synthesized in vitro in protein-synthesizing system programmed with human liver mRNA [Olofsson, S.-O., Elias, P., Bostrom, K., Lundholm, K., Norfeldt, P.-I., Wiklund, O., Fager, G., and Bondjers, G. (1983) 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 proteinsynthesizing 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 f 1.3 kDa (mean f SD, n = 12) could be precipitated. The observation that all monoclonal antibodies used reacted with a p~B~~~~ indicates a close immunological relation between this 80-kDa protein and apoB75kDd. Limited proteolysis of the 80-kDa protein (synthesized in the presence of [35S]-methionine) with Staphylococcus uureus V8 protease generated six [3'S]-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 This observation suggests a structural relation between the two proteins. Taken together, our results indicate that a protein corresponding to apoB'ISkDa is synthesized in vitro in a protein synthesizing system programmed with human liver mRNA (or total RNA fraction).We have also compared a p~B~~~~~ and the major component of apoLDL-2, apoBlOO [Kane, J. P., Hardman, D. A,, and Paulus, H. E. (1 980) Proc. Nut1 Acad. Sci USA 77,2465 -24691 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 apoBIOO and a p~B~~~"~, all reacted with a p~B~~~~~ and apoB100. These observations indicate a close immunological relation between the two proteins. Taken together our results support the hypothesis that apoBlOO has a subunit structure. We therefore suggest that apoB75kDa is a subunit of apoBlOO synthesized in human liver.

Pulse‐chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2

“…All antibodies used to precipitate apoB proteins from cell lysate or from culture medium were purified by adsorption chromatography as described earlier [20]. Immunoglobulins were isolated by ammonium sulphate precipitation [20].…”

“…For immunoprecipitation with monoclonal antibodies the samples were incubated for 4 h at room temperature with 100 Fg of non-immune mouse antibodies (in buffer B) (20), 150 pg of rabbit antibodies to mouse immunoglobulins (in buffer B) was then added and the incubation was continued for 2 h. Immunoprecipitin (0.2 ml of buffer C) was added and the sample was incubated for an additional hour. The Immunoprecipitin pellet was treated as described for the precipitation with polyclonal antibodies (see above).…”

“…In a separate experiment we measured the radioactivity recovered in apoB after 5,15,20,25,30,35,60 and 90 rnin chase. The results, shown in Fig.…”

“…We have previously presented data suggesting that an 80-kDa protein related to apoB could be precipitated with monoclonal antibodies to apoB from a human mRNA programmed in vitro protein-synthesizing system [20]. We have also isolated a 75-kDa protein from LDL-2 (referred to as a p~B~~ or apoB 14 [16, 201) that appears to correspond to the 80-kDa protein isolated from the in vitro translation system.…”

“…We suggested that these data support the hypothesis that apoB consists of proteins with relatively small molecular mass [9 -161. However, considering the problems involved in the interpretation of data from in vitro translation systems we have also pointed out that the isolated proteins may represent partial translation products of apoB [20] of the kind that has been described in other systems [21]. Since the studies in the in vitro translation system did not give unequivocal results, we turned to a whole cell system, the Hep G2 cells [22, 231. The pulse-chase studies presented in this paper demonstrate that apoB is synthesized in these cell as one polypeptide with a very large molecular mass.…”