Matthew J. Callow

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 apoBs 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.

Expression profiling and comparative sequence derived insights into lipid metabolism

Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes that are involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes that are involved in important processes in lipid metabolism such as sterol efflux, but also the utilization of information from these studies has added to our understanding of the regulation of pathways that participate in the process. Coupled with these gene expression studies, cross-species comparison (a technique used to search for sequences that are conserved through evolution) has proven to be a powerful tool to identify important noncoding regulatory sequences and novel genes that are relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (that which encodes apolipoprotein AV) that has dramatic effects on triglyceride metabolism in mice and humans.