Human lecithin:cholesterol acyltransferase (LCAT, E.G. 18.104.22.168) is a serine-type esterase that contains six cysteines, two of which, Cys31 and Cys184, are free. The remaining cysteines form disulfide links. One of these is between Cys50 and Cys74 and the other is between Cys313 and Cys356. The cDNA of LCAT and mutants in which one or two of the six cysteines were replaced by glycine was expressed in COS-6 cells. Polymerase chain reactions and Northern blot analysis indicated that LCAT mRNA was produced by all transfectants. Western blots of all transfected cells probed with a polyclonal antibody revealed intracellular LCAT. Substitution of glycine for either Cys50, Cys74, Cys313, or Cys356 was associated with a nearly total absence of activity in the medium. No protein was secreted when glycine replaced either of the amino acid residues that link Cys313 and Cys356. The small amounts of the Cys50 → Gly and Cys74 → Gly mutants found in the medium had specific activities that were much lower than that of the wild-type LCAT. All other transfectants secreted immunologically measurable amounts of active enzyme. Mutants in which one or both free cysteines, Cys31 and Cys184, were replaced with glycine were less active than the wild type and only partially inhibited by a sulfhydryl blocking reagent. The substrate specificities of the Cys31 → Gly and Cys184 → Gly mutants differed from that of the wild type. The specific activity of the wild-type LCAT against a substrate composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was 8 times higher than that measured against a substrate containing diarachidonoylphosphatidylcholine. The specific activities of mutants were nearly the same with both substrates. These data support a model of LCAT in which (1) Cys31 and Cys184 are not required for catalysis but sterically block access to the active-site region and (2) the disulfide bonds in native LCAT are required for activity and secretion-competent LCAT.
|Original language||English (US)|
|Number of pages||6|
|State||Published - Mar 1 1993|
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