Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties
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 carboxymethyla...
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doaj-849096fa55c649258b347e7472ca726e2021-04-26T05:46:18ZengElsevierJournal of Lipid Research0022-22751998-04-01394807820Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic propertiesAndras G. Lacko0Andrew J. Reason1Colin Nuckolls2Bhalchandra J. Kudchodkar3Maya P. Nair4Geetha Sundarrajan5P. Haydn Pritchard6Howard R. Morris7Anne Dell8To whom correspondence should be addressed.; Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London, U.K.Department of Chemistry, Columbia University, New York, NY 10027Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, TX 76107Department of Pathology, University of British Columbia, Vancouver, CanadaDepartment of Biochemistry, Imperial College of Science, Technology, and Medicine, London, U.K.Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London, U.K.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.http://www.sciencedirect.com/science/article/pii/S0022227520325682LCATglycoprotein structure |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Andras G. Lacko Andrew J. Reason Colin Nuckolls Bhalchandra J. Kudchodkar Maya P. Nair Geetha Sundarrajan P. Haydn Pritchard Howard R. Morris Anne Dell |
spellingShingle |
Andras G. Lacko Andrew J. Reason Colin Nuckolls Bhalchandra J. Kudchodkar Maya P. Nair Geetha Sundarrajan P. Haydn Pritchard Howard R. Morris Anne Dell Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties Journal of Lipid Research LCAT glycoprotein structure |
author_facet |
Andras G. Lacko Andrew J. Reason Colin Nuckolls Bhalchandra J. Kudchodkar Maya P. Nair Geetha Sundarrajan P. Haydn Pritchard Howard R. Morris Anne Dell |
author_sort |
Andras G. Lacko |
title |
Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties |
title_short |
Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties |
title_full |
Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties |
title_fullStr |
Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties |
title_full_unstemmed |
Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties |
title_sort |
characterization of recombinant human plasma lecithin: cholesterol acyltransferase (lcat): n-linked carbohydrate structures and catalytic properties |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
1998-04-01 |
description |
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. |
topic |
LCAT glycoprotein structure |
url |
http://www.sciencedirect.com/science/article/pii/S0022227520325682 |
work_keys_str_mv |
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