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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
Fonseca-Maldonado, Raquel [1] ; Vieira, Davi Serradella [2] ; Alponti, Juliana Sanchez [2] ; Bonneil, Eric [3] ; Thibault, Pierre [3] ; Ward, Richard John [2, 4]
Total Authors: 6
Affiliation:
[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
Total Affiliations: 4
Document type: Journal article
Source: Journal of Biological Chemistry; v. 288, n. 35, p. 25522-25534, AUG 30 2013.
Web of Science Citations: 31
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)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)

FAPESP's process: 10/18850-2 - Identification, characterization and engineering of plant cell wall degrading enzymes
Grantee:Richard John Ward
Support Opportunities: Research Projects - Thematic Grants
FAPESP's process: 08/57908-6 - National Institute of Science and Technology of Bioethanol
Grantee:Marcos Silveira Buckeridge
Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants