Site-specific mutagenesis demonstrates that cysteine 4326 of apolipoprotein B is required for covalent linkage with apolipoprotein (a) in vivo.
In the formation of the lipoprotein(a) (Lp(a)) particle, apolipoprotein(a) (apo(a)) and apolipoprotein B (apoB) are covalently linked via a disulfide bond in both humans and human-apo(a)/apoB transgenic mice. Studies based upon fluorescent labeling of free cysteine residues have suggested that cysteine 3734 of the 4 carboxyl-terminal cysteines of apoB (Cys-3734, Cys-3890, Cys-4190, and Cys-4326) is the most likely candidate to form a disulfide bond with apo(a). However, other recent studies using truncated apoB molecules suggest that Cys-4326, the terminal cysteine of apoB, may be implicated in the binding to apo(a). In order to definitively show which of apoB's carboxyl-terminal cysteines is essential in interacting with apo(a) we have used RecA-assisted restriction enzyme digestion coupled with site-specific mutagenesis to convert Cys-3734 and Cys-4326 to serine within separate 90-kilobase pair apoB P1 phagemid clones. Transgenic mice containing the normal or mutated apoB transgenes were created, and the covalent association of mutated apoB with apo(a) was assessed in mice transgenic for both apoB and apo(a). Analysis by ultracentrifugation and immunoblotting revealed that Cys-4326, but not Cys-3734, was essential in the formation of the covalent bond between apo(a) and apoB in vivo.
Callow
M J
MJ
Human Genome Center, Lawrence Berkeley Laboratory, University of California, Berkeley 94720, USA.
Rubin
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry / Q000378:metabolism*; D001055:Apolipoproteins B / Q000737:chemistry* / Q000378:metabolism*; D053241:Apoprotein(a); D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D017931:DNA Primers; D006801:Humans; D017270:Lipoprotein(a); D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D001053:Apolipoproteins; D001055:Apolipoproteins B; D017931:DNA Primers; D017270:Lipoprotein(a); D011994:Recombinant Proteins; D053241:Apoprotein(a); D003545:Cysteine