TY - JOUR
T1 - Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source
AU - Ayyobi, A. F.
AU - Lacko, A. G.
AU - Murray, K.
AU - Nair, M.
AU - Li, M.
AU - Molhuizen, H. O.F.
AU - Pritchard, P. H.
N1 - Funding Information:
We are grateful to Dr. John S. Hill for critical review of the manuscript. We also would like to thank Ms. Lida Adler and Sandra McGladdery for insightful discussions on LCAT assays and technical support. The carbohydrate analysis was performed by M-Scan Inc. This research was supported in part by the Heart and Stroke Foundation of British Columbia and Yukon, and a NATO Collaborative Research grant. H.O.F.M. was supported by a grant from the Netherlands Heart Foundation.
PY - 2000/2/24
Y1 - 2000/2/24
N2 - 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. (C) 2000 Elsevier Science B.V.
AB - 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. (C) 2000 Elsevier Science B.V.
KW - Carbohydrate structure
KW - Enzyme kinetics
KW - Lecithin:cholesterol acyltransferase
KW - Nuclear magnetic resonance
KW - Protein expression
KW - Reverse cholesterol transport
UR - http://www.scopus.com/inward/record.url?scp=0033953321&partnerID=8YFLogxK
U2 - 10.1016/S1388-1981(99)00199-7
DO - 10.1016/S1388-1981(99)00199-7
M3 - Article
C2 - 10685026
AN - SCOPUS:0033953321
SN - 1388-1981
VL - 1484
SP - 1
EP - 13
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 1
ER -