title: Modulation of the exocellular serine-thiol proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides.
abstract: Our group characterized an exocellular serine-thiol proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-thiol proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 microM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 microM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-thiol proteolytic activity, which is possibly involved in fungal dissemination.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, UNIFESP, Rua Botucatu, 862, oitavo andar, São Paulo, SP 04023-062, Brazil.
authors.forename: Alisson L
authors.initials: AL
authors.lastname: Matsuo
authors.forename: Ivarne I L
authors.initials: II
authors.lastname: Tersariol
authors.forename: Silvia I
authors.initials: SI
authors.lastname: Kobata
authors.forename: Luiz R
authors.initials: LR
authors.lastname: Travassos
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.forename: Rosana
authors.initials: R
authors.lastname: Puccia
mesh_terms: D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005029:Ethylenediamines / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010228:Paracoccidioides / Q000187:drug effects / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: C121956:2-aminobenzoylmethionyl-lysyl-alanyl-leucyl-threonyl-leucyl-glutamyldinitrophenylethylenediamine; D005029:Ethylenediamines; D006025:Glycosaminoglycans; D008351:Mannans; D009842:Oligopeptides; D003546:Cysteine Endopeptidases