Mapping N-linked glycosylation of carbohydrate-active enzymes in the secretome of Aspergillus nidulans grown on lignocellulose

Autor(es)

Rubio, Marcelo Ventura ; Zubieta, Mariane Paludetti ; Loureno Franco Cairo, Joao Paulo ; Calzado, Felipe ; Paes Leme, Adriana Franco ; Squina, Fabio Marcio ; Prade, Rolf Alexander ; de Lima Damasio, Andre Ricardo

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNOLOGY FOR BIOFUELS; v. 9, AUG 8 2016.
• Citações Web of Science: 8

Resumo

Background: The genus Aspergillus includes microorganisms that naturally degrade lignocellulosic biomass, secreting large amounts of carbohydrate-active enzymes (CAZymes) that characterize their saprophyte lifestyle. Aspergillus has the capacity to perform post-translational modifications (PTM), which provides an additional advantage for the use of these organisms as a host for the production of heterologous proteins. In this study, the N-linked glycosylation of CAZymes identified in the secretome of Aspergillus nidulans grown on lignocellulose was mapped. Results: Aspergillus nidulans was grown in glucose, xylan and pretreated sugarcane bagasse (SCB) for 96 h, after which glycoproteomics and glycomics were carried out on the extracellular proteins (secretome). A total of 265 proteins were identified, with 153, 210 and 182 proteins in the glucose, xylan and SCB substrates, respectively. CAZymes corresponded to more than 50 % of the total secretome in xylan and SCB. A total of 182 N-glycosylation sites were identified, of which 121 were detected in 67 CAZymes. A prevalence of the N-glyc sequon N-X-T (72.2 %) was observed in N-glyc sites compared with N-X-S (27.8 %). The amino acids flanking the validated N-glyc sites were mainly composed of hydrophobic and polar uncharged amino acids. Selected proteins were evaluated for conservation of the N-glyc sites in Aspergilli homologous proteins, but a pattern of conservation was not observed. A global analysis of N-glycans released from the proteins secreted by A. nidulans was also performed. While the proportion of N-glycans with Hex5 to Hex9 was similar in the xylan condition, a prevalence of Hex5 was observed in the SCB and glucose conditions. Conclusions: The most common and frequent N-glycosylated motifs, an overview of the N-glycosylation of the CAZymes and the number of mannoses found in N-glycans were analyzed. There are many bottlenecks in protein production by filamentous fungi, such as folding, transport by vesicles and secretion, but N-glycosylation in the correct context is a fundamental event for defining the high levels of secretion of target proteins. A comprehensive analysis of the protein glycosylation processes in A. nidulans will assist with a better understanding of glycoprotein structures, profiles, activities and functions. This knowledge can help in the optimization of heterologous expression and protein secretion in the fungal host. (AU)

• Processo FAPESP: 14/15403-6 - Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de "Unfolded Protein Response"
• Beneficiário: Mariane Paludetti Zubieta
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 14/06923-6 - Recalcitrância da biomassa de cana-de-açúcar: fundamentos relacionados à formação da parede celular, ao pré-tratamento e à digestão enzimática, aplicados no desenvolvimento de novos modelos de biorrefinarias
• Beneficiário: Andre Luis Ferraz
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Temático
• Processo FAPESP: 11/20977-3 - Investigação de vias de degradação de biomassa lignocelulósica no cupim Coptotermes gestroi complementares a hidrolases glicolíticas visando aplicação na produção de bioprodutos derivados de biomassa lignocelulósica
• Beneficiário: João Paulo Lourenço Franco Cairo
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/24988-5 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
• Beneficiário: Marcelo Ventura Rubio
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 12/20549-4 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
• Beneficiário: André Ricardo de Lima Damasio
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Jovens Pesquisadores
• Processo FAPESP: 14/23051-2 - Análise comparativa do mecanismo de secreção de proteínas heterólogas em Aspergillus nidulans
• Beneficiário: Felipe Calzado
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 09/54067-3 - EMU: aquisição de um espectrômetro de massas acoplado a cromatografia líquida para permitir ampliar a capacidade de atendimento de usuários e disponibilizar novas tecnologias no Laboratório de Espectrometria de Massas do Centro de Biologia Molecular Estrutural (ABTLUS)
• Beneficiário: Adriana Franco Paes Leme
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários