Glycosylation Of Alpha Research Articles

Production of β-Maltooligosaccharides of α- and δ-Tocopherols by Klebsiella pneumoniae and Cyclodextrin Glucanotransferase as Anti-Allergic Agents

The glycosylation of α- and δ-tocopherols using Klebsiella pneumoniae and cyclodextrin glucanotransferase (CGTase) was investigated. K. pneumoniae converted α- and δ-tocopherols into the corresponding β-glucosides in 10 and 8% yield, respectively. CGTase glycosylated α-tocopheryl β-glucoside to α-tocopheryl β-maltoside (51%) and α-tocopheryl β-maltotrioside (35%). On the other hand, δ-tocopheryl β-glucoside was converted into the corresponding β-maltoside (45%) and β-maltotrioside (29%) by CGTase. The β-glucoside of α-tocopherol, and β-glucoside and β-maltoside of δ-tocopherol showed inhibitory effects on IgE antibody production and on histamine release from rat peritoneal mast cells.

Global and site‐specific detection of human integrin α5β1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions. Glycomics studies most often aim at identifying glycosylation sites, and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation. In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides, or glycans must be isolated from glycopeptide fractions and analyzed as pools. This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site. A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used. Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology. The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program. The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC). A specific collected fraction was then analyzed by MS before and after glycan release. These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo.

The influence of alpha1-acid glycoprotein on collagenase-3 activity in early rheumatoid arthritis.

The concentration and glycosylation of alpha(1)-acid glycoprotein (AGP) alter significantly during inflammation. A definitive physiological role for AGP remains elusive and is the subject of extensive investigation. This study investigated the influence of AGP on the activity of collagenase-3, an important mediator of cartilage destruction in rheumatoid arthritis. AGP was isolated from normal and rheumatoid plasma. Fucosylation was determined by high pH anion-exchange chromatography; sialylation was assessed following enzymatic digest. Rheumatoid AGP displayed elevated fucosylation and sialylation compared with normal. The influence of each sample on collagenase-3 activity was measured fluorometrically. AGP influenced collagenase-3 catalysis and collagen binding, with catalytic activity correlating with fucosylation. Rheumatoid AGP exhibited less efficient inhibition than normal plasma AGP. It is hypothesized that AGP within rheumatoid synovial fluid may be inadequate to prevent excessive cartilage destruction and hence may exacerbate the disease process.

The structure of the oligosaccharides of alpha3beta1 integrin from human ureter epithelium (HCV29) cell line.

There is a growing line of evidence that glycosylation of alpha and beta subunits is important for the function of integrins. Integrin alpha3beta1, from human ureter epithelium cell-line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin alpha3beta1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.

Implications of the invariant gamma-chain on the intracellular transport of class II histocompatibility antigens.

We have examined the role of the invariant gamma-chain on the intracellular transport of human class II histocompatibility antigens. mRNA was selected by hybridization to cDNA corresponding to class II alpha-, beta-, and gamma-chains, and the obtained mRNA fractions were injected individually and in various combinations into X. laevis oocytes. Translation products were isolated after various periods of chase, and their carbohydrate moieties were analyzed to monitor the subcellular localization of polypeptide chains. A mixture of alpha-, beta-, and gamma-chains, or gamma-chains alone, were transported and glycosylated to the same extent as in a B lymphoblastoid cell line. However, although alpha- and beta-chains formed a complex in the absence of the gamma-chain, the transport of this complex was slowed down. Furthermore, the glycosylation of alpha- and beta-chains appeared incomplete. Thus, the invariant gamma-chain seems to play a crucial role for the rate of transport and glycosylation of class II alpha- and beta-chains.