Substrate and positional specificities of human and mouse lecithin-cholesterol acyltransferases. Studies with wild type recombinant and chimeric enzymes expressed in vitro.
Human lecithin-cholesterol acyltransferase (LCAT) preferentially attacks sn-1 position of 16:0-20:4 phosphatidylcholine (PC), producing more 16:0 cholesteryl ester (CE) than 20:4 CE. In contrast, rat and mouse LCATs produce mostly 20:4 CE from the same PC. To understand the structural basis for this difference in positional specificity, we studied the specificities of recombinant mouse and human LCATs and several chimeric constructs of the two. The rLCATs retained the substrate and positional specificities of the plasma enzymes when expressed in COS-1 cells. Human and mouse LCAT cDNAs were each cleaved into three fragments, recombined in various combinations, and the chimeric products were analyzed for their specificities. When the N-terminal, or (and) C-terminal segments of human LCAT were replaced by the corresponding mouse LCAT segments, the chimeric products exhibited the specificity of intact human enzyme. However, when the middle segment, containing the residues 130-306 was replaced by the corresponding mouse LCAT segment, the enzyme exhibited the specificity of mouse LCAT. Similarly, the mouse rLCAT exhibited the specificity of human enzyme when its central segment, but not its N-terminal or C-terminal segment was replaced by the corresponding segment from human LCAT. These results show that the substrate and positional specificities of LCAT are controlled by the central domain of LCAT protein, corresponding to the amino acid residues 130-306.
Subbaiah
P V
PV
Department of Medicine, Rush Medical College, Chicago, IL 60612.
Liu
M
M
Senz
J
J
Wang
X
X
Pritchard
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002678:Chimera; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase