title: Glycan modification of a thermostable recombinant (1-3, 1-4)-beta-glucanase secreted from Saccharomyces cerevisiae is determined by strain and culture conditions.
abstract: High level biosynthesis and secretion of the thermostable hybrid (1-3, 1-4)- beta-glucanase H(A16-M) has been achieved in Saccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1P) and the Bacillus macerans (1-3, 1-4)-beta-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3'-phosphoglycerate kinase promoter, each in two different Saccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y.
mesh_terms: D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003470:Culture Media; D004795:Enzyme Stability; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006031:Glycosylation; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D013045:Species Specificity
chemical_list: D003470:Culture Media; D011134:Polysaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase