Proteome-Wide Analysis of Single-Nucleotide Variations in the N-Glycosylation Sequon of Human Genes

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can result in the gain of glycosylation. Although the general importance of glycosylation is well known and acknowledged, the effect of variation on the actual glycoproteome of an organism is still mostly unknown. In this study, we focus on a comprehensive analysis of non-synonymous single nucleotide variations (nsSNV) that lead to either loss or gain of the N-glycosylation motif. We find that 1091 proteins have modified N-glycosylation sequons due to nsSNVs in the genome. Based on analysis of proteins that have a solved 3D structure at the site of variation, we find that 48% of the variations that lead to changes in glycosylation sites occur at the loop and bend regions of the proteins. Pathway and function enrichment analysis show that a significant number of proteins that gained or lost the glycosylation motif are involved in kinase activity, immune response, and blood coagulation. A structure-function analysis of a blood coagulation protein, antithrombin III and a protease, cathepsin D, showcases how a comprehensive study followed by structural analysis can help better understand the functional impact of the nsSNVs

FACTOR-V LEIDEN GENE MUTATION AND THROMBIN GENERATION IN RELATION TO THE DEVELOPMENT OF ACUTE STROKE

To determine the prevalence of the factor V Leiden gene mutation in relation to the phenotypes of cerebral infarction and cerebral hemorrhage, we studied 386 randomly selected cases of acute stroke and 247 control subjects. Factor V genotype was determined by amplification of a 267-bp sequence of exon/intron 10 of the factor V gene. Levels of prothrombin fragment F-1+2, a marker of thrombin generation, were determined in both acute and convalescent stroke and related to factor V genotype. Prothrombin fragment F-1+2, was assessed by using an enzyme-linked immunosorbent assay. Sixteen stroke cases (4.1%) were identified as having the mutation compared with 14 (5.6%) control subjects. Prothrombin fragment F-1+2, levels were estimated in 191 cases and found to be elevated both acutely and after 3 months, but they were not related to factor V genotype. Prothrombin fragment F-1+2 is elevated in acute stroke and requires further evaluation in relation to cerebrovascular disease. These results suggest that the factor V Leiden gene mutation is not a risk factor for arterial thrombosis causing stroke

Identification of antithrombin-modulating genes. Role of LARGE, a gene encoding a bifunctional

The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteinsThis study was supported partially by 04515/GERM/06 (Fundación Séneca de la Región de Murcia), SAF2009-08993 and SAF2008/01859 (Spanish Ministerio de Ciencia y Tecnología & Fondo Europeo de Desarrollo Regional de la Unión Europea FEDER), PI-08/0756, PI-11/0184 and RECAVA RD06/0014/0039 & RD06/0014/0016 (Spanish Instituto de Salud Carlos III & Fondo Europeo de Desarrollo Regional de la Unión Europea FEDER), and Centre National du Genotypage (Evry, France). MEMB is a holder of a predoctoral research grant from Spanish Instituto de Salud Carlos III (FI09/00190). IMM is a researcher from Fundació n para la Formación e Investigación Sanitarias. JNF is a postdoctoral researcher of the University of Murcia. JM Soria was supported by ‘‘Programa d’ Estabilització d’Investigadors de la Direcció d’Estrategia i Coordinació del Departament de Salut’’ (Generalitat de Catalunya)