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 ser ine residues, including Ser181, which is thought to be part of the cat alytic site. In order to determine the importance of these serine resi dues 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 adjac ent asparagine residues in two additional mutants, LCAT (Ser19 --> Ala , Asn20 --> Thr) and LCAT (Ser383 --> Ala, Asn384 --> Thr), in order t o ascertain the effect of the serines on N-glycosylation. The mutant c omplementary DNA (cDNA) were subcloned into a eukaryotic expression ve ctor (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-speci fic bands in media and lysates of the transfected cells. With two exce ptions, the amounts of LCAT mass secreted by the transfectants were si milar to that of the wild type (mean, 90% mass of wild type; range, 34 -138%). Except for LCAT (Ser181 --> Gly), which was inactive, the spec ific activities of the remainder of the mutant enzymes were also simil ar (mean, 95% activity of wild type; range, 65-169%), These results in dicate that Ser181 is part of the catalytic site and that stereoconser vative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.