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Paracoccin from Paracoccidioides brasiliensis ; purification through affinity with chitin and identification of N ‐acetyl‐β‐ D ‐glucosaminidase activity
Author(s) -
dos Reis Almeida Fausto Bruno,
de Oliveira Leandro Licursi,
de Sousa Marcelo Valle,
Barreira Maria Cristina Roque,
Hanna Ebert Seixas
Publication year - 2010
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1731
Subject(s) - paracoccidioides brasiliensis , paracoccidioidomycosis , biology , dimorphic fungus , chitin , paracoccidioides , western blot , biochemistry , mannan , lectin , microbiology and biotechnology , cell wall , affinity chromatography , yeast , enzyme , polysaccharide , chitosan , gene
Abstract The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N ‐acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFα and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the β‐1,4‐homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin‐affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS–PAGE and Western blot analysis revealed similarities between the N ‐acetylglucosamine‐ and chitin‐bound fractions, confirmed by MALDI–TOF–MS of trypsinic peptides. Western blot of two‐dimensional gel electrophoresis of the yeast extract showed a major spot with M r 70 000 and pI approximately 5.63. Morevover, an N ‐acetyl‐β‐ D ‐glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright © 2009 John Wiley & Sons, Ltd.