Dataset Information

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

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.

SUBMITTER: Tie JK

PROVIDER: S-EPMC3956053 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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

Tie Jian-Ke JK Zheng Mei-Yan MY Pope R Marshall RM Straight David L DL Stafford Darrel W DW

Biochemistry 20061201 49

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 carb ...[more]

PMID: 17144668

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Project description:The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners.

Project description:The addition of asparagine (N)-linked polysaccharide chains (i.e., glycans) to the gp120 and gp41 glycoproteins of human immunodeficiency virus type 1 (HIV-1) envelope is not only required for correct protein folding, but also may provide protection against neutralizing antibodies as a "glycan shield." As a result, strong host-specific selection is frequently associated with codon positions where nonsynonymous substitutions can create or disrupt potential N-linked glycosylation sites (PNGSs). Moreover, empirical data suggest that the individual contribution of PNGSs to the neutralization sensitivity or infectivity of HIV-1 may be critically dependent on the presence or absence of other PNGSs in the envelope sequence. Here we evaluate how glycan-glycan interactions have shaped the evolution of HIV-1 envelope sequences by analyzing the distribution of PNGSs in a large-sequence alignment. Using a "covarion"-type phylogenetic model, we find that the rates at which individual PNGSs are gained or lost vary significantly over time, suggesting that the selective advantage of having a PNGS may depend on the presence or absence of other PNGSs in the sequence. Consequently, we identify specific interactions between PNGSs in the alignment using a new paired-character phylogenetic model of evolution, and a Bayesian graphical model. Despite the fundamental differences between these two methods, several interactions are jointly identified by both. Mapping these interactions onto a structural model of HIV-1 gp120 reveals that negative (exclusive) interactions occur significantly more often between colocalized glycans, while positive (inclusive) interactions are restricted to more distant glycans. Our results imply that the adaptive repertoire of alternative configurations in the HIV-1 glycan shield is limited by functional interactions between the N-linked glycans. This represents a potential vulnerability of rapidly evolving HIV-1 populations that may provide useful glycan-based targets for neutralizing antibodies.

Dataset Information

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

Publications

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

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