Effects of mutations in Aedes aegypti sterol carrier protein-2 on the biological function of the protein.
Sterol carrier protein-2 (SCP-2) is a nonspecific intracellular lipid carrier protein. However, the molecular mechanism of ligand selectivity and the in vivo function of SCP-2 remain unclear. In this study, we used site-directed mutagenesis to investigate the ligand selectivity and in vivo function of the yellow fever mosquito sterol carrier protein-2 protein (AeSCP-2). Mutations to amino acids in AeSCP-2 known to interact with bound ligand also weakened NBD-cholesterol binding. Substitution of amino acids in the ligand cavity changed the ligand specificity of mutant AeSCP-2. Overexpressing wild-type AeSCP-2 in the Aedes aegypti cultured Aag-2 cells resulted in an increase in the level of incorporation of [(3)H]cholesterol. However, overexpressing mutants that were deleterious to the binding of NBD-cholesterol in AeSCP-2 showed a loss of ability to enhance uptake of [(3)H]cholesterol in cultured cells. Interestingly, when [(3)H]palmitic acid was used as the substrate for incorporation in vivo, there was no change in the levels of incorporation with overexpression of wild-type protein or mutated AeSCP-2s. The in vivo data suggest that AeSCP-2 is involved in sterol uptake, but not fatty acid uptake. This is the first report that the cholesterol binding ability may directly correlate with AeSCP-2's in vivo function in aiding the uptake of cholesterol.
Radek
James T
JT
Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Dyer
David H
DH
Lan
Que
Q
D000330:Aedes / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D002352:Carrier Proteins; D008024:Ligands; C072087:sterol carrier proteins; D002784:Cholesterol