Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source.
Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.
Ayyobi
A F
AF
Atherosclerosis Specialty Laboratory, Department of Pathology and Laboratory Medicine, University of British Columbia, St. Paul's Hospital, 1081 Burrard Street, Suite 180-20, Vancouver, B.C., Canada.
Lacko
A G
AG
Murray
K
K
Nair
M
M
Li
M
M
Molhuizen
H O
HO
Pritchard
P H
PH
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D006031:Glycosylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009005:Monosaccharides / Q000032:analysis; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000096:biosynthesis* / Q000737:chemistry; D010957:Plasmids; D011134:Polysaccharides / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D014162:Transfection; D014407:Tumor Cells, Cultured
D009005:Monosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase