Effects of site-directed mutagenesis on the serine residues of human lecithin:cholesterol acyltransferase.
Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19-->Ala), LCAT (Ser181-->Gly), LCAT (Ser208-->Ala), LCAT (SEr216-->Ala), LCAT (Ser225-->Ala) and LCAT (Ser383-->Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19-->Ala, Asn20-->Thr) and LCAT (Ser383-->Ala, Asn384-->Thr), in order to ascertain the effect of the serines on N-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34-138%). Except for LCAT (Ser181-->Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean 95% activity of wild type; range, 65-169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.
Qu
S J
SJ
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Fan
H Z
HZ
Blanco-Vaca
F
F
Pownall
H J
HJ
D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018014:Gene Transfer Techniques; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012694:Serine / Q000737:chemistry*
D018076:DNA, Complementary; D012333:RNA, Messenger; D012694:Serine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase