Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

Author(s)

dos Reis Almeida, Fausto Bruno ^[1] ; de Oliveira, Leandro Licursi ^{[1, 2]} ; de Sousa, Marcelo Valle ^[3] ; Roque Barreira, Maria Cristina ^[1] ; Hanna, Ebert Seixas ^{[1, 4]}

Affiliation

^[1] Univ Sao Paulo, Dept Biol Celular & Mol & Bioagentes Patogen, Fac Med Ribeirao Preto, BR-14049900 Ribeirao Preto, SP - Brazil

^[2] Univ Fed Vicosa, Dept Biol Geral, BR-36570000 Minas Gerais - Brazil

^[3] Univ Brasilia, Ctr Brasileiro Serv & Pesquisa Prot, Inst Biol, BR-70910900 Brasilia, DF - Brazil

^[4] Univ Sao Paulo, Invent Biotecnol Ltda ME Incubadora Supera, BR-14040900 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: YEAST; v. 27, n. 2, p. 67-76, FEB 2010.
• Web of Science Citations: 16

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 or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-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 or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil 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) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-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 (C) 2009 John Wiley \& Sons. Ltd. (AU)

• FAPESP's process: 07/55731-9 - Biochemical characterization of paracoccin from Paracoccidioides brasiliensis
• Grantee: Fausto Bruno dos Reis Almeida
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 06/60642-2 - Lectins: biological effects and pharmaceutical applications
• Grantee: Maria Cristina Roque Antunes Barreira
• Support Opportunities: Research Projects - Thematic Grants