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 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
AG
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth 76107, USA.
Reason
A J
AJ
Nuckolls
C
C
Kudchodkar
B J
BJ
Nair
M P
MP
Sundarrajan
G
G
Pritchard
P H
PH
Morris
H R
HR
Dell
A
A
D016632:Apolipoprotein A-I / Q000378:metabolism; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics; D010740:Phospholipases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D016339:Spectrometry, Mass, Fast Atom Bombardment
D016632:Apolipoprotein A-I; D006023:Glycoproteins; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D010740:Phospholipases