title: Role of N-linked glycosylation of lecithin:cholesterol acyltransferase in lipoprotein substrate specificity.
abstract: Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of cholesteryl ester in plasma. LCAT is a glycoprotein which has a carbohydrate content estimated to be approx. 25% of its total mass. Previous studies of recombinant LCAT have characterized the function of the four N-linked glycosylation sites of LCAT with respect to reconstituted HDL analogue substrates. In order to investigate the relationship between N-linked glycosylation and the ability of LCAT to esterify cholesterol in native plasma lipoproteins, we have expressed a series of mutant LCAT cDNAs in which each of the four glycosylation consensus sequences was eliminated individually. All mutant LCAT proteins were secreted by stably transfected baby hamster kidney cells. The ability of mutant LCATs to esterify cholesterol in purified native lipoproteins indicated that the elimination of the carbohydrate chain at position 20 of recombinant LCAT was associated with a lower activity than the wild type enzyme when HDL was used as a substrate, but no inhibitory effect was observed when LDL was used as a substrate. A mutant enzyme with a substitution of Asn-84-->Gln or Asn-272-->Gln displayed a decreased ability to esterify cholesterol in either HDL or LDL. In contrast, the loss of a carbohydrate chain at position 384 was associated with an increase in enzyme activity for both HDL (1.5-fold) and LDL (2.5-fold) substrates. Kinetic analysis of these recombinant enzymes indicated that the apparent Km values for cholesterol in either HDL or LDL were not affected, but that the differences in activities were due to changes in the apparent Vmax. Heat inactivation studies were performed to assess the role of specific carbohydrate groups in enzyme stability. Loss of a carbohydrate chain at position 20, 272 or 384 decreased thermostability of LCAT whereas a mutation at position 84 did not affect thermostability. These results suggest that individual carbohydrate chains confer specific structural and functional properties to LCAT.
authors: Department of Pathology, University of British Columbia, Vancouver, Canada.
authors: K
authors: K
authors: O
authors: J S
authors: JS
authors: Hill
authors: P H
authors: PH
authors: Pritchard
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D008076:Cholesterol, HDL / Q000378:metabolism; D008078:Cholesterol, LDL / Q000378:metabolism; D016384:Consensus Sequence; D006224:Cricetinae; D004795:Enzyme Stability; D004951:Esterification; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D007668:Kidney; D008074:Lipoproteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D008074:Lipoproteins; D011994:Recombinant Proteins; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase