Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe

PLoS One. 2013 Jun 25;8(6):e66853. doi: 10.1371/journal.pone.0066853. Print 2013.

Abstract

α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Wall / metabolism
  • Gene Expression
  • Glucans / chemistry
  • Glucans / metabolism
  • Glycoside Hydrolases / genetics*
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism*
  • Mutation
  • Paracoccidioides / cytology
  • Paracoccidioides / enzymology*
  • Paracoccidioides / genetics
  • Schizosaccharomyces / genetics*
  • Sequence Alignment
  • Solubility
  • Transcription, Genetic

Substances

  • Glucans
  • alpha-1,3-glucan
  • Glycoside Hydrolases
  • exo-1,3-alpha-glucanase

Grants and funding

This work was partially supported by Project ICGEB-VEN05 of the International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. HV-D was recipient of a Ph.D scholarship awarded by FONACIT (Fondo Nacional de Ciencia, Tecnología e Innovación) Caracas, Venezuela. MP was recipiente of a MSc scholarship awarded by IVIC, Caracas Venezuela. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.