Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Autor(es)

Fonseca-Maldonado, Raquel ^[1] ; Vieira, Davi Serradella ^[2] ; Alponti, Juliana Sanchez ^[2] ; Bonneil, Eric ^[3] ; Thibault, Pierre ^[3] ; Ward, Richard John ^{[2, 4]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, BR-14049900 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14049901 Sao Paulo - Brazil

^[3] Univ Montreal, Inst Res Immunol & Canc, Montreal, PQ - Canada

^[4] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biological Chemistry; v. 288, n. 35, p. 25522-25534, AUG 30 2013.
• Citações Web of Science: 31

Resumo

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)GlcNAc(2) modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex(9)GlcNAc(2) 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. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 10/52322-3 - Bioprospecção de fungos filamentosos produtores de holoenzimas com aplicação em biorefinaria
• Beneficiário: Maria de Lourdes Teixeira de Moraes Polizeli
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOTA - Regular
• Processo FAPESP: 08/57908-6 - Instituto Nacional de Biotecnologia para o Bioetanol
• Beneficiário: Marcos Silveira Buckeridge
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Temático
• Processo FAPESP: 09/50838-5 - Determinantes de estabilidade estrutural de enzimas xilanases termofílicas mutantes por dinâmica molecular: efeitos da glicosilação sobre a termoestabilidade
• Beneficiário: Davi Serradella Vieira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado