Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity.
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 TNFalpha 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 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. 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.
dos Reis Almeida
Fausto Bruno
FB
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil.
de Oliveira
Leandro Licursi
LL
Valle de Sousa
Marcelo
M
Roque Barreira
Maria Cristina
MC
Hanna
Ebert Seixas
ES
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000908:Antibodies, Fungal / Q000276:immunology; D015153:Blotting, Western; D002473:Cell Wall / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D010228:Paracoccidioides / Q000201:enzymology / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000908:Antibodies, Fungal; D005656:Fungal Proteins; D037102:Lectins; D014409:Tumor Necrosis Factor-alpha; C548066:paracoccin, Paracoccidioides brasiliensis; D002686:Chitin; D009569:Nitric Oxide; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine