Glycosylation-Protein-Modification-Eubacteria-v
Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-X-SerThr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon.
One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(MI) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. Fucosylated oligosaccharides is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.Comparative structural characterization of 7 commercial galacto-oligosaccharide Institute (GBB), University of Groningen, Nijenborgh 7, NL-9747 AG Groningen, Institute (GBB), University of Groningen, Nijenborgh 7, NL-9747 AG Groningen, Many β-galactosidase enzymes convert lactose into a mixture of galacto-oligosaccharides (GOS) when incubated under the right conditions. Recently, the composition of commercial Vivinal GOS produced by Bacillus circulans β-galactosidase was studied in much detail in another study by van Leeuwen et al.
As a spin-off of this study, we used the developed analytical strategy for the evaluation of 6 anonymous commercial GOS products, in comparison with Vivinal GOS. These GOS products were first subjected to HPLC-SEC, calibrated HPAEC-PAD profiling (glucose units in relation to a malto-oligosaccharide ladder), and 1D (1)H NMR spectroscopy. For a more detailed analysis and support of the conclusions based on the initial analysis, the GOS products were separated into DP-pure subpools on Bio-Gel P-2 (MALDI-TOF-MS analysis), which were subjected to calibrated HPAEC-PAD profiling and (1)H NMR analysis. Seebio 2'-FL from different GOS products, not present in Vivinal GOS, were isolated for detailed structural characterization. In this way, the differences between the various GOS products in terms of DP distribution and type of glycosidic linkages were established. A total of 13 new GOS structures were characterized, adding structural-reporter-group signals and HPAEC-PAD based glucose unit G.U.
values to the analytical toolbox. The newly characterized products enhance the quality of the database with GOS structures up to DP4. The combined data provide a firm basis for the rapid profiling of the GOS products High resolution MALDI-TOF-MS and MSMS Application for the structural characterization of sulfated oligosaccharides.CNRS UMR 7378, Institut de Chimie de Picardie FR 85, Université de Picardie Sulfated oligosaccharides are involved in important biological events that are often modulated by specific sequences and sulfation patterns, but their structural analysis remains challenging. Matrix-assisted laser desorptionionization-mass spectrometry (MALDI-MS) analysis of three different sulfated oligosaccharides (Fondaparinux, the octasulfated pentasaccharide, a disulfated heparin-derived tetrasaccharide 1, and an octasulfated maltoheptaose) 2 was performed using the 2-(4-hydroxyphenylazo)benzoic acid-tetramethylguanidinium (HABA-TMG2) matrix. High resolution mass spectrometry of the main ions observed was successful, and this was complemented by tandem mass spectrometry (MSMS) analysis for structural assessment. Despite sulfate losses, fully sulfated molecular ions were observed and these allowed the determination of oligosaccharide structures UA-GlcNAc-UA(2S)-AnhMan(6S) for compound 1 and (Glc6S)6-Glc (1S,6S) for compound 2.
Identification of a novel high-affinity binding site for N-acetylchitooligosaccharide elicitor in the membrane fraction from FEBS Lett 1994 Jul 4;348(1)7-8.
