Abstract
The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide 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 introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).
Highlights
The structural features of apolipoprotein B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood
To document that the apoB95 mutation had been retained within the pop-in Yeast Artificial Chromosome (YAC), we enzymatically amplified a segment of the apoB gene flanking the apoB95 nonsense mutation and cleaved the amplified DNA with SpeI (Fig. 2B, lane 2); for the apoB97 experiments, the polymerase chain reaction (PCR) product was cleaved with StuI (Fig. 2B, lane 4)
We have been interested in defining the structure of Lp(a), with an emphasis on defining the structural features of apoB that are required for its interaction with apo(a)
Summary
The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. ApoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB’s initial interaction with apo(a) and for facilitating the formation of the disulfide bond. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a) These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331– 4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, residues 4331– 4397, is necessary for the efficient assembly of Lp(a). The simplest hypothesis regarding the noncovalent interaction event would be that apo(a) binds to apoB sequences near the critical free cysteine residue, cysteine 4326 In considering this hypothesis, we examined the amino acid sequences surrounding residue 4326, both for human and mouse apoB. These observations led us to hypothesize that the sequences carboxylterminal from cysteine 4326 might play a role in Lp(a) assembly
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