Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).
Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.
D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism*; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D053241:Apoprotein(a); D001483:Base Sequence; D001665:Binding Sites; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004220:Disulfides; D005998:Glycine; D006801:Humans; D017270:Lipoprotein(a); D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping
D001053:Apolipoproteins; D001055:Apolipoproteins B; D017931:DNA Primers; D004220:Disulfides; D017270:Lipoprotein(a); D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D053241:Apoprotein(a); D003545:Cysteine; D005998:Glycine