Purification and subcellular localization of a secreted 75 kDa Trypanosoma cruzi serine oligopeptidase.

An extracellular serine peptidase was purified 460-fold from Trypanosoma cruzi epimastigotes culture supernatant with (NH(4))(2)SO(4) precipitation followed by affinity chromatography aprotinin-agarose and continuous elution electrophoresis, yielding a total recovery of 65%. The molecular mass of the active enzyme estimated by reducing and non-reducing SDS-PAGE was about 75kDa. The optimal pH and temperature of this glycosylated peptidase were 8.0 and 37 degrees C using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. The enzyme did not hydrolyze polypeptide substrates but was active against short peptide substrates containing arginine at the P1 site, in both ester and amide bonds. The peptidase was inhibited by TPCK and TCLK but not by other protease inhibitors suggesting that the enzyme belongs to the serine peptidase class. Interestingly, the enzyme seems to demonstrate some metal dependence since its activity was reduced by 1,10-phenanthroline, calcium and zinc ions. Rabbit anti-T. cruzi extracellular serine peptidase antiserum was used to show that the enzyme was restricted to intracellular structures, including the flagellar pocket, plasma membrane and cytoplasmic vesicles resembling reservosomes. These results suggest that the serine oligopeptidase is secreted into the extracellular environment through the flagellar pocket and the intracellular location could suggest its participation in certain proteolysis events in reservosomes. These findings show that this peptidase is a novel T. cruzi serine oligopeptidase, which differs not only from other peptidases described in the same parasite but also in other species of Trypanosoma.

Oligopeptidase B from Leishmania amazonensis: molecular cloning, gene expression analysis and molecular model.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. A complete open reading frame of oligopeptidase B from Leishmania amazonensis was amplified with polymerase chain reaction with gradient annealing temperatures using primers designed for the oligopeptidase B gene from L. major. The 2,196-bp fragment coded for a protein of 731 amino acids with a predicted molecular mass of 83.49 KDa. The encoded protein (La_OpB) shares a 90% identity with oligopeptidases of L. major and L. infantum, 84% with L. braziliensis, and approximately 62% identity with Trypanosoma peptidases. The oligopeptidase B gene is expressed in all cycle stages of L. amazonensis. The three dimensional model of La_OpB was obtained by homology modeling based on the structure of prolyl oligopeptidases. We mapped a La_OpB model that presents a greater negative charge than prolyl oligopeptidases; our results suggest a difference in the S2 subsite when compared to oligopeptidases B from Trypanosoma and bacterial oligopeptidases B. The La_OpB model serves as a starting point for its exploration as a potential target source for a rational chemotherapy.

Oligopeptidase B from L. amazonensis: molecular cloning, gene expression analysis and molecular model.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. A complete open reading frame of oligopeptidase B from Leishmania amazonensis was amplified with polymerase chain reaction with gradient annealing temperatures using primers designed for the oligopeptidase B gene from L. major. The 2,196-bp fragment coded for a protein of 731 amino acids with a predicted molecular mass of 83.49 KDa. The encoded protein (La_OpB) shares a 90% identity with oligopeptidases of L. major and L. infantum, 84% with L. braziliensis, and approximately 62 identity with Trypanosoma peptidases. The oligopeptidase B gene is expressed in all cycle stages of L. amazonensis. The three dimensional model of La_OpB was obtained by homology modeling based on the structure of prolyl oligopeptidases. We mapped a La_OpB model that presents a greater negative charge than prolyl oligopeptidases; our results suggest a difference in the S2 subsite when compared to oligopeptidases B from Trypanosoma and bacterial oligopeptidases B. The La_OpB model serves as a starting point for its exploration as a potential target source for a rational chemotherapy.

BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease.

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.

Extracellular metalloproteinase activity in Phytomonas françai.

Extracellular proteolytic activities were detected in Phytomonas françai culture supernatant. A 67-kDa enzyme was purified by ammonium sulfate precipitation and gel filtration in a HPLC system. This proteinase was optimally active at 28 degrees C and pH 5.0; and the use of proteolytic inhibitors indicated that it belongs to the metalloproteinase class. This is the first report on the purification of an extracellular metalloproteinase from a Phytomonas species.

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells.

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.

Aspartic proteinase in Dugesia tigrina (Girard) planaria.

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease.

Identification and properties of two extracellular proteases from Brevundimonas diminuta

Extracellular proteases from Brevundimonas diminuta (syn. Pseudomonas diminuta) were studied in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) containing a copolymerized substrate. Two proteases were detected migrating at 67kDa and 50 kDa: both of them hydrolysed preferentially gelatin, but casein was also degraded and a slight hydrolysis was observed with hemoglobin. No detectable extracellular proteolytic activity was found in bovine serum albumin-containing gels. The optima temperature and pH for proteolytic activity were between 40ºC and 50ºC in a pH ranging from 7.0 to 11.0, respectively. These enzymes were isolated by analytical high performance liquid chromatography (HPLC). Proteases assays with the synthetic substrate Z-Phe-Arg-MCA and inhibitors EGTA, EDTA and 1, 10 phenanthroline point out that these enzymes are metalloproteases.