J. Lipid Res.
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The Journal of Lipid Research, Vol. 39, 807-820, April 1998
Copyright © 1998 by Lipid Research, Inc.

Original Article

Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties

Andras G. Lackoa, Andrew J. Reasonb, Colin Nuckollsc, Bhalchandra J. Kudchodkara, Maya P. Naira, Geetha Sundarrajana, P. Haydn Pritchardd, Howard R. Morrisb, and Anne Dellb
a Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107
b Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London, U.K.
c Department of Chemistry, Columbia University, New York, NY 10027
d Department of Pathology, University of British Columbia, Vancouver, Canada

Correspondence to: Andras G. Lacko.

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak® cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical.

These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.—Lacko, A. G., A. J. Reason, C. Nuckolls, B. J. Kudchodkar, M. P. Nair, G. Sundarrajan, P. H. Pritchard, H. R. Morris, and A. Dell. Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. J. Lipid Res. 1998. 39: 807–820.

Supplementary key words: LCAT, glycoprotein structure


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 J. Biol. Chem.Home page
J. Kosman and A. Jonas
Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase
J. Biol. Chem., September 28, 2001; 276(40): 37230 - 37236.
[Abstract] [Full Text] [PDF]


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