Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

Jian-Ke Tie; Mei-Yan Zheng; R Marshall Pope; David L Straight; Darrel W Stafford

doi:10.1021/bi0618518

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Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

Journal article

Peer reviewed

Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

Jian-Ke Tie, Mei-Yan Zheng, R Marshall Pope, David L Straight and Darrel W Stafford

Biochemistry (Easton), Vol.45(49), pp.14755-14763

12/12/2006

DOI: 10.1021/bi0618518

PMCID: PMC3956053

PMID: 17144668

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Abstract

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.

Amino Acid Sequence

Animals

Binding Sites

Carbon-Carbon Ligases - chemistry

Carbon-Carbon Ligases - metabolism

Escherichia coli

Glycosylation

Humans

Insecta

Kinetics

Models, Molecular

Molecular Sequence Data

Mutagenesis

Oligopeptides - chemistry

Oligosaccharides - chemistry

Oligosaccharides - isolation & purification

Peptide Fragments

Protein Conformation

Recombinant Proteins - chemistry

Recombinant Proteins - metabolism

Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Details

Title: Subtitle: Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function
Creators: Jian-Ke Tie - Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA
Mei-Yan Zheng
R Marshall Pope
David L Straight
Darrel W Stafford
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.45(49), pp.14755-14763
DOI: 10.1021/bi0618518
PMID: 17144668
PMCID: PMC3956053
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: HL48318 / NHLBI NIH HHS R01 HL048318 / NHLBI NIH HHS
Language: English
Date published: 12/12/2006
Academic Unit: Medicine Administration
Record Identifier: 9984632131302771

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