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

Author(s)

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

• Document type: Journal article
• Source: BIOTECHNOLOGY FOR BIOFUELS; v. 9, AUG 8 2016.
• Web of Science Citations: 8

Abstract

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)

• FAPESP's process: 14/15403-6 - Aspergillus nidulans as a model to manipulate unfolded protein response-related genes
• Grantee: Mariane Paludetti Zubieta
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/06923-6 - Sugar cane biomass recalcitrance: basic knowledge related to the cell wall construction, pretreatment and enzymatic digestion, applied for the development of innovative biorefinery models
• Grantee: Andre Luis Ferraz
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 11/20977-3 - Investigation of plant biomass conversion in termite Coptotermes gestroi complementary to glycosyl hydrolases aiming at bioproducts formation derived from lignocellulosic biomass.
• Grantee: João Paulo Lourenço Franco Cairo
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/24988-5 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases
• Grantee: Marcelo Ventura Rubio
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 12/20549-4 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases
• Grantee: André Ricardo de Lima Damasio
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
• FAPESP's process: 14/23051-2 - Comparative analysis of the heterologous protein secretion mechanisms in Aspergillus nidulans
• Grantee: Felipe Calzado
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 09/54067-3 - Acquisition of a mass spectrometer coupled to a liquid chromatography system for increasing the capacity to meet the needs of users and for making new technologies available in the Laboratory of Mass Spectrometry
• Grantee: Adriana Franco Paes Leme
• Support Opportunities: Multi-user Equipment Program