Effects of site-directed mutagenesis on the serine residues of human lecithin: Cholesterol acyltransferase

Shi Jing Qu, Hui Zhen Fan, Francisco Blanco-Vaca, Henry J. Pownall

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

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 Ser 181, 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.

Original languageEnglish (US)
Pages (from-to)803-809
Number of pages7
JournalLipids
Volume29
Issue number12
DOIs
StatePublished - Dec 1994

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Cell Biology

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