Changes In Glycosylation State Research Articles

Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

Change of fucosylated IgG2 Fc-glycoforms in pancreatitis and pancreatic adenocarcinoma: a promising disease-classification model

The fixed constant (Fc) region of IgG is subject to changes in glycosylation state in response to diseases. On the basis of sera from 75 healthy controls, 75 pancreatitis (PT) patients, and 75 pancreatic adenocarcinoma (PAC) patients, we analyzed six fucosylated glycoforms of IgG2 (G0F, G1F, G2F, G0FN, G1FN, and G2FN), by matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS), to evaluate their use as biomarkers for pancreatic diseases. Compared with healthy controls, significant increases in agalactosylated glycoforms and decreases in galactosylated glycoforms were observed for PT and PAC patients. Logistic regression analysis suggested that truncation of the sugar chain was prone to occur in PT and, especially, PAC patients. After participants were stratified by sex and age, receiver operating characteristic curve analysis revealed good overall sensitivity and specificity for discrimination of PAC and PT patients from healthy controls. A combination of G0F and galactosylation also had acceptable power for differentiating PAC patients from PT patients.

Fine Epitope Mapping of Monoclonal Antibody 5F1 Reveals Anticatalytic Activity toward the N Domain of Human Angiotensin-Converting Enzyme

Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.