title: Engineering the pattern of protein glycosylation modulates the thermostability of a GH11 xylanase.
abstract: Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex(8-16)GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.
authors.affiliation: From the Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo CEP 14049-900, Brazil.
authors.forename: Raquel
authors.initials: R
authors.lastname: Fonseca-Maldonado
authors.affiliation: the Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo CEP 14049-901, Brazil.
authors.forename: Davi Serradella
authors.initials: DS
authors.lastname: Vieira
authors.affiliation: the Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo CEP 14049-901, Brazil.
authors.forename: Juliana Sanchez
authors.initials: JS
authors.lastname: Alponti
authors.affiliation: the Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada, and.
authors.forename: Eric
authors.initials: E
authors.lastname: Bonneil
authors.affiliation: the Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada, and.
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
authors.affiliation: the Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo CEP 14049-901, Brazil,; the Laboratório Nacional de Ciência e Tecnologia do Bioetanol/Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo CEP 13083-970, Brazil. Electronic address: rjward@ffclrp.usp.br.
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006358:Hot Temperature; D016296:Mutagenesis; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Glycoside Hydrolases; Glycosylation; Mass Spectrometry (MS); Molecular Dynamics Simulation; Protein Design; Protein Engineering; Protein Thermostability