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

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-...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Héctor Villalobos-Duno, Gioconda San-Blas, Maryan Paulinkevicius, Yolanda Sánchez-Martín, Gustavo Nino-Vega
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/580d22219bfc452e8b5bcd49d50db63c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:580d22219bfc452e8b5bcd49d50db63c
record_format	dspace
spelling	oai:doaj.org-article:580d22219bfc452e8b5bcd49d50db63c2021-11-18T07:40:09ZBiochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.1932-620310.1371/journal.pone.0066853https://doaj.org/article/580d22219bfc452e8b5bcd49d50db63c2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23825576/?tool=EBIhttps://doaj.org/toc/1932-6203α-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.Héctor Villalobos-DunoGioconda San-BlasMaryan PaulinkeviciusYolanda Sánchez-MartínGustavo Nino-VegaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e66853 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Héctor Villalobos-Duno Gioconda San-Blas Maryan Paulinkevicius Yolanda Sánchez-Martín Gustavo Nino-Vega Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
description	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.
format	article
author	Héctor Villalobos-Duno Gioconda San-Blas Maryan Paulinkevicius Yolanda Sánchez-Martín Gustavo Nino-Vega
author_facet	Héctor Villalobos-Duno Gioconda San-Blas Maryan Paulinkevicius Yolanda Sánchez-Martín Gustavo Nino-Vega
author_sort	Héctor Villalobos-Duno
title	Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
title_short	Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
title_full	Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
title_fullStr	Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
title_full_unstemmed	Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous Expression in Schizosaccharomyces pombe.
title_sort	biochemical characterization of paracoccidioides brasiliensis α-1,3-glucanase agn1p, and its functionality by heterologous expression in schizosaccharomyces pombe.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/580d22219bfc452e8b5bcd49d50db63c
work_keys_str_mv	AT hectorvillalobosduno biochemicalcharacterizationofparacoccidioidesbrasiliensisa13glucanaseagn1panditsfunctionalitybyheterologousexpressioninschizosaccharomycespombe AT giocondasanblas biochemicalcharacterizationofparacoccidioidesbrasiliensisa13glucanaseagn1panditsfunctionalitybyheterologousexpressioninschizosaccharomycespombe AT maryanpaulinkevicius biochemicalcharacterizationofparacoccidioidesbrasiliensisa13glucanaseagn1panditsfunctionalitybyheterologousexpressioninschizosaccharomycespombe AT yolandasanchezmartin biochemicalcharacterizationofparacoccidioidesbrasiliensisa13glucanaseagn1panditsfunctionalitybyheterologousexpressioninschizosaccharomycespombe AT gustavoninovega biochemicalcharacterizationofparacoccidioidesbrasiliensisa13glucanaseagn1panditsfunctionalitybyheterologousexpressioninschizosaccharomycespombe
_version_	1718423097822937088

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

Ejemplares similares