We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.A01Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Carmona, Adriana Karaoglanovic

Juliano, Luiz

Juliano, Maria Aparecida

Puccia, Rosana

Travassos, Luiz Rodolpho

Brazilian Journal of Medical and Biological Research

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.A01Universidade Federal de São Paulo (UNIFESP)UNIFESP, EPM, São Paulo, BrazilSciEL

Puccia, Rosana [UNIFESP]

Juliano, Maria Aparecida [UNIFESP]

Juliano, Luiz [UNIFESP]

Travassos, Luiz Rodolpho [UNIFESP]

Carmona, Adriana Karaoglanovic [UNIFESP]

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme

R. Puccia

M.A. Juliano

L. Juliano

L.R. Travassos

A.K. Carmona

Directory of Open Access Journals

Repositório Institucional UNIFESP

645Braz J Med Biol Res 32(5) 1999 P. brasiliensis serine-thiol proteinaseBrazilian Journal of Medical and Biological Research (1999) 32: 645-649ISSN 0100-879XDetection of the basementmembrane-degrading proteolyticactivity of Paracoccidioides brasiliensisafter SDS-PAGE using agarose overlayscontaining Abz-MKALTLQ-EDDnpDepartamentos de 1Microbiologia, Imunologia e Parasitologia and2Biofísica, Universidade Federal de São Paulo, São Paulo, SP, BrasilR. Puccia1, M.A. Juliano2,L. Juliano2, L.R. Travassos1and A.K. Carmona2AbstractWe have characterized, in the Paracoccidioides brasiliensis yeastphase, an exocellular SH-dependent serine proteinase activity againstAbz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides,and showed that it is also active against constituents of the basementmembrane in vitro. In the present study, we separated the componentsof P. brasiliensis culture filtrates by electrophoresis and demonstratedthat the serine-thiol exocellular proteinase has a diffuse and heteroge-neous migration by SDS-PAGE, localizing in a region between 69 and43 kDa. The hydrolytic activity was demonstrable after SDS-PAGEusing buffered agarose overlays of Abz-MKALTLQ-EDDnp, follow-ing incubation at 37oC, and detection of fluorescent bands with a UVtransilluminator. Hydrolysis was more intense when incubation wascarried out at basic pH, and was completely inhibited with 2.5 mMPMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mMp-HMB), suggesting its serine-thiol nature. A proteolytic band withsimilar characteristics was observed in conventional gelatin zymo-grams, but could not be correlated with a silver-stained component.Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the base-ment membrane degrading enzyme.CorrespondenceR. PucciaDepartamento de Microbiologia,Imunologia e Parasitologia, UNIFESPRua Botucatu 862, 8º andar04023-062 São Paulo, SPBrasilFax: +55-11-571-5877E-mail: rosana.dmip@epm.brPresented at the 5th BrazilianSymposium on ExtracellularMatrix - SIMEC, Angra dos Reis,RJ, Brasil, September 7-10, 1998.Research supported by FAPESP(No. 1995/0559-8), BID-FINEP,Pronex and CNPq.Received November 17, 1998Accepted December 21, 1998Key words· P. brasiliensis· Serine-thiol proteinase· SDS-PAGE· Fluorescent-quenchedpeptidesParacoccidioides brasiliensis is a dimor-phic fungus that causes human paracoccidi-oidomycosis (PCM), a systemic mycosis en-demic in Latin America, which can be fatalin immunocompromised individuals if notsuccessfully treated. Patients with PCM canhave multiple clinical forms which are de-pendent on the relationship between the hostsresistance mechanisms and fungal pathoge-nicity (1). The yeast cell wall a-glucan (2)and the gp43 antigen (3,4), which is protec-tive in experimental murine PCM (5), havebeen implicated in fungal virulence. Othervirulence factors in P. brasiliensis probablyexist.We have characterized a subtilisin-like,646Braz J Med Biol Res 32(5) 1999R. Puccia et al.SH-dependent serine proteinase activity inP. brasiliensis grown in the yeast phase (6).Internally quenched fluorescent peptidesderived from MKRLTL and flanked by Abz(ortho-aminobenzoyl) and EDDnp (ethyl-enediaminedinitrophenyl) were cleaved atthe L/T bond when incubated with a P. bra-siliensis culture filtrate. The exocellular pro-teinase activity had an optimum pH of >9.0and was irreversibly inhibited by PMSF,mercuric acetate and sodium 7-hydroxymer-curibenzoate (p-HMB). Moreover, the en-zyme was able to selectively cleave, in vitro,components of the basal membrane or asso-ciated with it, including laminin, fibronec-tin, type IV collagen and proteoglycans. Suchactivity could be of biological significancein tissue invasion by the fungus (7).The serine-thiol proteolytic activity in-volves an enzyme of very high specific activ-ity, which is rather unstable and is lost afterseveral chromatographic steps. So far, a singlesilver-stainable band in SDS-PAGE has notbeen observed in fractionated preparations,which has hampered the purification andcharacterization of the enzyme. The objec-tive of the present study was to detect theexocellular serine-thiol proteinase activityin SDS-PAGE gels using overlays of Abz-MKALTLQ-EDDnp. This is a substrateanalogous to Abz-MKRLTL-EDDnp but witha better Vmax/Km value (6), and was synthe-sized as described previously (8,9). A con-ventional gelatin zymogram was used in par-allel.P. brasiliensis strain 339 (originally pro-vided by Dr. Angela Restrepo-Moreno,Medellin, Colombia) was cultivated at 35oC,with shaking, in modified YPD (0.5% Bacto-yeast extract, 0.5% casein peptone and 1.5%glucose), and the culture supernatant wascollected by paper filtration at the peak ofproteinase activity against Abz-MKALTLQ-EDDnp (7). Ammonium sulfate was addedto the culture filtrate at 40% saturation andthe precipitate separated by centrifugation(16300 g, 30 min). The supernatant (50 ml)was concentrated by FPLC in a PhenylSuperose HR 5/5 (Pharmacia/LKB, Uppsala,Sweden) column, according to standard pro-cedures. The proteolytic activity against Abz-MKALTLQ-EDDnp was concentrated in twofractions, denoted F3 and F4, which wereanalyzed on substrate gels. This concentra-tion procedure using Phenyl Superose, al-though more laborious, resulted in a betteryield of the active enzyme than precipitationof culture filtrates with 50% ammonium sul-fate (6), since very little of the hydrolyticactivity is precipitated with salt.For SDS-PAGE analysis, F3 and F4 wereelectrophoresed (100 V, at 4oC) in conven-tional linear 10% gels (10), using samplebuffer containing SDS at 0.5% final concen-tration without boiling. Pre-stained low mo-lecular weight protein markers (Gibco BRL,Gaithersburg, MD, USA) were applied tothe gels, and their migration position wasmarked with a needle after running as areference for localization of the proteolyticbands. The gels were then washed for onehour in 0.05 M Tris-HCl, pH 8.5, 10% MeOH(11), with three changes, and finally in buf-fer without MeOH. Abz-MKALTLQ-EDDnp(100 µg/ml) overlays were prepared in 1%buffered agarose (0.75 mm thick), placed onthe washed SDS-PAGE gels, and incubatedat 37oC in a moist chamber. Fluorescentgreen bands were visualized with a UV trans-illuminator (302 nm) and the results wererecorded on Polaroid films using a yellowfilter.Figure 1A shows the fluorescence ob-tained after increasing incubation times withAbz-MKALTLQ-EDDnp in agarose over-lays. The proteolytic activity appeared as adiffuse zone in the gel between bovine se-rum albumin (BSA) and egg ovalbumin(OVA), or between 69 and 43 kDa apparentmolecular masses. The fluorescent band at43 kDa (gp43) seen in F4 was not due toproteolysis, but to natural fluorescence emit-ted by the molecule, and could still be seenwithout the peptide overlay, or when the647Braz J Med Biol Res 32(5) 1999 P. brasiliensis serine-thiol proteinasesample was previously boiled in SDS forprotease inactivation (data not shown). Theprotein profiles of F3 and F4 (Figure 2A)indicated that F4 was rich in the gp43 com-ponent, which could barely be seen in F3 bysilver staining. This fractionation after Phe-nyl Superose chromatography has been ad-vantageous in that only F3 fractions are nowbeing processed for proteinase purification.This procedure avoids the gp43 depletionstep by affinity chromatography which wasused before (6). On the other hand, F3 isusually twice more active than F4 againstAbz-MKALTLQ-EDDnp in solution.In the experiment shown in Figure 1A,maximum fluorescence arising from the hy-drolyzed substrate was achieved after 1 hand 45 min of incubation at 37oC. After 3 h,it diffused and was not as intense. The fluo-rescence intensity and the incubation timenecessary to reach a maximum of fluores-cence varied with the activity of the samplein a dose-dependent fashion (data not shown).The result shown in Figure 1A was similarwhen the samples were incubated with re-duced or nonreduced SDS-PAGE samplebuffer.The specificity of the activity against Abz-MKALTLQ-EDDnp in agarose overlays wasassessed by pre-incubation (30 min, roomtemperature) of F3 with known protease in-hibitors before addition of the sample bufferand electrophoresis. The result is illustratedin Figure 1B, where proteolysis was com-pletely inactivated by 2.5 mM PMSF (lane4), and by boiling in SDS buffer (lane 2); p-HMB (0.15 and 2.5 mM) was partially inhib-itory, especially at higher concentrations (lane7*). Since this is not an active site inhibitor,it could have been partly dissociated duringelectrophoresis. In addition, the activity wasmore intense upon incubation at pH 8.5 thanat pH 6.5 (data not shown). These resultsstrongly suggest that the exocellular serine-thiol activity previously characterized in P.brasiliensis (6) is due to a proteinase whichmigrates diffusely between 69 and 43 kDa inB- 69- 431 2 3 4 5 6 7 7*Figure 1 - Demonstration of P. brasiliensis exocellular proteolytic activity in SDS-PAGE gelsusing Abz-MKALTLQ-EDDnp in agarose overlays, and enzyme characterization with pro-tease inhibitors. A, The hydrolytic activity in F3 (1.25 µg) and F4 (5.0 µg) was recorded after45 min, 1 h and 45 min, and 3 h of incubation at 37oC. Fluorescence of the 43-kDacomponent was intrinsic, and was observed without the peptide overlay. B, F3 (1.25 µg)alone (lane 1), or inactivated by boiling (lane 2), pre-incubated with 2.5 mM ortho-phenan-throline and 5 mM EDTA (lane 3), 2.5 mM PMSF (lane 4), 0.13 mM trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, E-64 (lane 5), 2.5% DMSO (lane 6), 0.15 mM (lane 7) and2.5 mM (lane 7*) p-HMB. Molecular mass markers (kDa) are indicated on the right side.F3 F43 ho/nF3 F4Figure 2 - Silver-stained SDS-PAGE (A) and zymogram (B) profiles of F3 and F4 fractions of P.brasiliensis exocellular components. For the zymograms, the polyacrylamide gels wereimpregnated with 0.1% gelatin. After the electrophoretic run, the gels were washed withbuffered MeOH, as indicated in the text, and incubated at 37oC in 50 mM Tris-HCl, pH 8.5,for 3 h or overnight (o/n), as indicated. Proteolytic bands were visualized by staining withCoomassie brilliant blue. The amounts loaded onto the gels were: 0.5 µg of F3 and 1 µg ofF4 for silver staining and 5 µg of F4, 1.25 µg (3 h) or 0.25 µg (o/n) of F3 for zymograms.Molecular weight markers (kDa) are indicated on the right side.- 106- 69- 43- 69- 43- 69- 43A BA45 min1 h 45 min3 h- 69- 43- 43- 69- 69- 43F3 F4648Braz J Med Biol Res 32(5) 1999R. Puccia et al.SDS-PAGE gels. This is also in accordancewith our previous data (6) showing that theexocellular serine-thiol activity against Abz-MKRLTL-EDDnp was eluted in a singlepeak near ovalbumin. Furthermore, the dif-fuse SDS-PAGE migration of the proteinaseis probably due to heterogeneous and/or abun-dant glycosylation, as inferred from bindingof the enzymatic activity to concanavalin A(Puccia R and Carmona AK, unpublishedresults).The same results were obtained when F3and F4 were analyzed for gelatinase activityin a zymogram (Figure 2B), where a fuzzyproteolytic band between 69 and 43 kDacould be seen even after 3 h of incubation at37oC. After overnight incubation, the pro-teolytic zone was very clear and broad. Theproteolysis inhibition pattern and the pHdependence in zymograms were similar tothose with Abz-MKALTLQ-EDDnp in aga-rose overlays. Additional results obtainedwith gelatin zymograms showed that theserine-thiol proteinase activity was not al-tered by heating at 45oC or by lyophilization,and could not be detected after incubation atroom temperature (data not shown). Ther-mostability was checked in protein-richpreparations due to the unstable nature of theproteinase in more purified fractions.Gelatinase activity in culture filtrates ofP. brasiliensis concentrated by heat evapo-References1. Franco M (1987). Host-parasite relation-ships in paracoccidioidomycosis. Journalof Medical and Veterinary Mycology, 25:5-18.2. San-Blas G & San-Blas F (1994). Biochem-istry of Paracoccidioides brasiliensis di-morphism. In: Franco M, Lacaz CS,Restrepo-Moreno A & Del Negro C (Edi-tors), Paracoccidioidomycosis. CRC Press,Boca Raton.3. Vicentini AP, Gesztesi JL, Franco MF,Souza W, Moraes JZ, Travassos LR &Lopes JD (1994). Binding of Paracoccidi-oides brasiliensis to laminin through sur-face glycoprotein gp43 leads to enhance-ment of fungal pathogenesis. Infectionand Immunity, 62: 1465-1469.4. Gesztesi JL, Puccia R, Travassos LR,Vicentini AP, Franco MF & Lopes JD(1996). Monoclonal antibodies against the43,000 Da glycoprotein from Paracoccidi-oides brasiliensis modulate laminin-medi-ated fungal adhesion to epithelial cellsand pathogenesis. Hybridoma, 15: 415-422.5. Taborda CP, Juliano MA, Puccia R, FrancoM & Travassos LR (1998). Mapping of theT-cell epitope in the major 43-kilodaltonglycoprotein of Paracoccidioides brasilien-sis which induces a Th-1 response protec-tive against fungal infection in BALB/cmice. Infection and Immunity, 66: 786-793.6. Carmona AK, Puccia R, Oliveira MCF,Rodrigues E, Juliano L & Travassos LR(1995). Characterization of an exocellularserine-thiol proteinase activity in Paracoc-cidioides brasiliensis. Biochemical Jour-nal, 30: 209-214.7. Puccia R, Carmona AK, Gesztesi JL,Juliano L & Travassos LR (1998). Exocel-lular proteolytic activity of Paracoccidi-oides brasiliensis: cleavage of compo-nents associated with the basementmembrane. Medical Mycology, 36: 345-348.8. Chagas JR, Juliano L & Prado ES (1991).Intramolecularly quenched fluorogenictetrapeptide substrates for tissue andration at 45oC and lyophilization has beenreported (12). For strain 339, the authorsshowed what seems to be two overlappingbroad bands between 43 and 78 kDa. Thisgelatinase activity may have been due to theserine-thiol proteinase, on the basis of simi-larity in molecular weight and heat-stability.However, this is probably not the case, sincethat activity was very strong upon incubationat room temperature. However, it is unlikelythat the gelatinase activity mentioned above(12) was due to gp43, as attributed by theauthors without any evidence, since therewere a number of components migrating inthe same broad region of the proteolyticzone. Moreover, protease motifs have notbeen reported in the gp43 gene sequence(13) and the proteolytic properties associ-ated with gp43 in the past (14) were latershown to be due to aggregation (6).In the present work, we show for the firsttime the usefulness of internally quenchedfluorescent peptides flanked by Abz (ortho-aminobenzoyl) and EDDnp (ethylenedi-aminedinitrophenyl) in agarose overlays forthe detection of proteolytic activities afterSDS-PAGE. For P. brasiliensis serine-thiolproteinase, visualization of the activity zonein substrate gels is essential for further char-acterization and purification of the basementmembrane degrading enzyme.649Braz J Med Biol Res 32(5) 1999 P. brasiliensis serine-thiol proteinaseplasma kallikreins. Analytical Biochemis-try, 192: 419-425.9. Oliveira MCF, Hirata IY, Chagas JR,Boschcov P, Gomes RAS, Figueiredo AFS& Juliano L (1992). Intramolecularlyquenched fluorogenic peptide substratesfor human renin. Analytical Biochemistry,203: 39-46.10. Laemmli UK (1970). Cleavage of struc-tural proteins during the assembly of thehead of bacteriophage T4. Nature, 227:680-685.11. Lundy FT, Magee AC, Blair IS & McDwellDA (1995). A new method for detectionof proteolytic activity in Pseudomonaslundensis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Elec-trophoresis, 16: 43-45.12. Vaz CAC, Mackenzie DWR, Hearn V,Camargo ZP, Singer-Vermes LM, BurgerE & Calich VLG (1994). Gelatinase activityof exoantigens from virulent and non-viru-lent isolates of Paracoccidioides brasilien-sis. Journal of Medical and Veterinary My-cology, 32: 65-69.13. Cisalpino PS, Puccia R, Yamauchi LM,Cano MIN, Silveira JF & Travassos LR(1996). Cloning and characterization of thegene encoding the major diagnostic anti-gen of Paracoccidioides brasiliensis: ex-pression of epitopes of the 43,000 Daglycoprotein recognized by antibodiesfrom human patients. Journal of Biologi-cal Chemistry, 271: 4553-4560.14. Puccia R & Travassos LR (1991). The 43-kDa glycoprotein from Paracoccidioidesbrasiliensis and its deglycosylated form:excretion and susceptibility to proteoly-sis. Archives of Biochemistry and Bio-physics, 289: 298-302.

Puccia, R.

Juliano, M.A.

Juliano, L.

Travassos, L.R.

Carmona, A.K.

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Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

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