TRANSGENIC MICE EXPRESSING HUMAN APOB95 AND APOB97

The structural features of apolipoprotein (apo) B that are important f or its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are i ncompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside f rom a single free cysteine residue, must be important for apoB's initi al interaction with apo(a) and far facilitating the formation of the d isulfide bond, To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used ''pop-in, pop out'' gene targeting in a human apoB yeast artificial chromosome to in troduce nonsense mutations into exon 29 of the apoB gene, The mutant y east artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutan t forms of apoB in transgenic mice, As judged by in vitro assays of Ly (a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a ) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Ep( a) rapidly, although not quite as rapidly as the full-length apoB100 ( 4536 amino acids). These results were supported by an analysis of doub le-transgenic mice expressing both human apo(a) and either-apoB95 or a poB97. Iu mice expressing bath apoB95 and apo(a), there was only a tra ce amount of Lp(a) in the plasma, and most of the apo(a) was free, whe reas in mice expressing both apoB97 and apo(a), virtually all of the a po(a) was bound to apoB97 in the farm of Lp(a), These results show tha t sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are no t absolutely required for Lp(a) formation, but this segment of the apo B molecule, particularly residues 4331-4397, is necessary for the effi cient assembly of LP(a).