Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis

Abstract

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

C-Npys (S-3-nitro-2-pyridinesulfenyl) and peptide derivatives can inhibit a serine-thiol proteinase activity from Paracoccidioides brasiliensis

“…1, bottom). Enzymatic cleavage of Abz-MKALTLQ-EDDnp or human fibronectin was due to PbST, since hydrolysis was specifically inhibited by p-HMB and PMSF (not shown), as previously reported [2]. In our experimental conditions, the activity against Abz-MKALTLQ-EDDnp was fully bound to the column and was recovered in the eluted fractions at a yield of about 65%.…”

“…In previous publications we characterized the PbST activity using preparations of P. brasiliensis supernatants enriched by either ion exchange/gel-filtration chromatography [2,3] or hydrophobic phenyl-Superose columns [4,17]. To date, we have tried to purify the proteinase using many different chromatographic methods [not published], however total purification of the active proteinase has never been achieved due to aggregation to other fungal components and/or loss of enzymatic activity after several chromatographic steps.…”

“…The proteolytic activity of PbST against Abz-MKALTLQ-EDDnp was determined at 37°C in 50 mM Tris-HCl, pH 8.0, in a Hitachi F-2000 spectrofluorometer (excitation=320 nm; emission=420 nm), as described previously [2]. The effect of Boc-C(Npys), Bzl-MKRLTLC(Npys)-NH 2 and Bzl-C(Npys)KRLTL-NH 2 in the enzymatic activity was determined using the same procedure, after 5 min of pre-incubation with the enzyme preparation.…”

“…Our group has previously characterized an extracellular subtilisin-like serine proteinase activity in the yeast phase of the human pathogen Paracoccidioides brasiliensis [2]. This activity is able to selectively degrade, in vitro, murine laminin, human fibronectin, type IVcollagen, and proteoglycans, while common proteins like bovine serum albumin (BSA) or casein are not cleavable substrates [3].…”

“…The proteinase is able to hydrolyze fluorescence resonance energy (FRET) peptides homologous to MKRLTL, which are flanked by Abz (ortho-aminobenzoic acid) and EDDnp (N- [2,4-dinitrophenyl]ethylenediamine). Cleavage occurs between the Leu-Thr bond at an optimum alkaline pH and is inhibited by PMSF, mercuric acetate, and p-HMB (sodium 7-hydroxymercuribenzoate), but not by E-64 [2]. Therefore, the enzymatic activity has been classified as a thiol-containing subtilisin-like serine proteinase (PbST, for P. brasiliensis serine-thiol proteinase) belonging to the group of proteinase K. In addition, we have recently described a novel type of regulation of the PbST activity against Abz-MKALTLQ-EDDnp by neutral polysaccharides, including a fungal extracellular galactomannan, which might help stabilize the enzyme [4].…”