Produção Nacional
Revisado por pares
Cysteine Proteinase Activity Regulation
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30433
ISSN1083-351X
AutoresPaulo C. Almeida, Iseli L. Nantes, Cláudia C.A. Rizzi, Wagner Alves de Souza Júdice, Jair R. Chagas, Luiz Juliano, Helena B. Nader, Ivarne L.S. Tersariol,
Tópico(s)Enzyme Production and Characterization
ResumoPapain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the α-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced α-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce α-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH. Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the α-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced α-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce α-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH. 1-[[(l-trans-epoxysuccinyl)-l-leucyl]amino]-4-guanidino-butane carbobenzoxyl-l-phenylalanyl-l-arginine-4-methylcoumarinyl-7-amide ortho-aminobenzoyl-l-alanyl-l-phenylalanyl-l-arginyl-l-seryl-l-seryl-l-alanyl-l-glutamineN-(ethylenediamine)-2,4-dinitrophenylamide high pressure liquid chromatography Among the sulfated glycosaminoglycans, heparan sulfate, a ubiquitous cell surface component of animals cells, exhibits the highest structural variability according to the tissue and species of origin (1Dietrich C.P. Nader H.B. Straus H.A. Biochem. Biophys. Res. Commun. 1983; 111: 865-871Crossref PubMed Scopus (94) Google Scholar, 2Dietrich C.P. Tersariol I.L.S. Toma L. Moraes C.T. Porcionatto M.A. Oliveira F.W. Nader H.B. Cell. Mol. Biol. 1998; 44: 417-429PubMed Google Scholar, 3Nader H.B. Dietrich C.P. Heparin: Chemical and Biological Properties, Clinical Applications. CRC Press Inc., Boca Raton, FL1989Google Scholar). Most cellular heparan sulfate, at the cell surface and in extracellular matrix, derives from the syndecan proteoglycan family (4Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar). These classes of compounds are heteropolysaccharides composed of several distinct disaccharides containing uronic acid and glucosamine with N- and 6-O-sulfates and N-acetyl substitutions. The interaction of heparan sulfate and heparin with proteins regulates a broad spectrum of biological processes. These proteins fall into quite diverse groups, such as proteins involved in hemostasis, proteins of extracellular matrix, growth factors, proteins of lipid metabolism, and others (5Conrad H.E. Heparin-Binding Proteins. Academic Press Inc., New York1998Google Scholar). It has been shown that heparin can modify the activities of some serine proteinases and its natural inhibitors in vitro (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology. R. G. Landes Co., Austin, TX1996Google Scholar, 7Frommherz K.J. Faller B. Bieth J.G. J. Biol. Chem. 1991; 266: 15356-15362Abstract Full Text PDF PubMed Google Scholar, 8Ermolieff J. Boudier C. Laine A. Meyer B. Bieth J.G. J. Biol. Chem. 1994; 269: 29502-29508Abstract Full Text PDF PubMed Google Scholar) and also that heparan sulfate proteoglycans, syndecan-1 ectodomain, and syndecan-4 ectodomain are shed into acute inflammatory wound fluids (9Subramanian S. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). The purified syndecan-1 ectodomain protects cathepsin G from inhibition by α1-antichymotrypsin and squamous cell carcinoma antigen 2, and it protects elastase from inhibition by α1-proteinase inhibitor. Moreover, the degradation of endogenous heparan sulfate from wound fluids reduces proteolytic activities in the fluid. These results strongly suggest that syndecan-1 and syndecan-4 maintain the proteolytic balance in acute wound fluid (10Kainulainen V. Wang H. Schick C. Bernfield M. J. Biol. Chem. 1998; 273: 11563-11569Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Syndecans, via their heparan sulfate chain, bind many of the factors that orchestrate the inflammatory response to tissue injury, as well as a variety of extracellular matrix components (9Subramanian S. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 10Kainulainen V. Wang H. Schick C. Bernfield M. J. Biol. Chem. 1998; 273: 11563-11569Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Papain is considered to be the archetype of cysteine proteinases. The papain-like cysteine proteinases are the most abundant among the cysteine proteinases. This family consists of papain and related plant proteinases, such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, and the lysosomal cathepsins B, H, L, S, C, and K (11Turk B. Turk V. Turk D. Biol. Chem. 1997; 378: 141-150PubMed Google Scholar). The lysosomal cysteine proteinases cathepsins B and L have been implicated in a variety of pathological conditions, especially in diseases involving tissue remodeling states, such as tumor metastasis (12Iacobuzio-Donahue C.A. Shuja S. Cai J. Peng P. Murnane M.J. J. Biol. Chem. 1997; 272: 29190-29199Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Sloane B.F. Rozhin J. Johnson K. Taylor H. Crissman J.D. Honn K.V. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2483-2487Crossref PubMed Scopus (210) Google Scholar), parasite infection (14Del Nery E. Juliano M.A. Lima A.P.C.A. Scharfstein J. Juliano L. J. Biol. Chem. 1997; 272: 25713-25718Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Lalmanach G. Lecaile F. Chagas J.R. Authié E. Scharfstein J. Juliano M.A. Gauthier F. J. Biol. Chem. 1998; 273: 25112-25116Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), arthritis (16Mort J.S. Recklies A.D. Poole A.R. Arthrits Rheum. 1984; 27: 509-515Crossref PubMed Scopus (124) Google Scholar), and other types of inflammatory injury (17Katunuma N. Intracellular Proteolysis. Japan Scientific Societies Press, Tokyo1989Google Scholar). Cathepsins B and L can participate in metastasis formation by degradation of several extracellular matrix components (18Sheahan K. Shuja S. Murnane M.J. Cancer Res. 1989; 49: 3809-3814PubMed Google Scholar, 19Buck M.R. Karutis D.G. Day N.A. Honn K.V. Sloane B.F. Biochem. J. 1992; 282: 273-278Crossref PubMed Scopus (368) Google Scholar, 20Barret A.J. Kirschke H. Methods Enzymol. 1981; 80: 535-561Crossref PubMed Scopus (1729) Google Scholar). Binding of cysteine proteinases with basement membranes is of significant interest for understanding the biological role of cysteine proteinases in tumor invasion and other types of tissue remodeling (21Guinec N. Dalet-Fumeron V. Pagano M. FEBS Lett. 1992; 24: 305-308Crossref Scopus (13) Google Scholar). Confocal microscopy image analysis indicated that cathepsin B was associated with the external basal cell surface in the murine B16 amelanotic melanoma cells (22Moin K. Cao L. Day N.A. Kobliski J.E. Sloane B.F. Biol. Chem. Hoppe-Seyler. 1998; 379: 1093-1099Crossref PubMed Scopus (35) Google Scholar). It has been shown that membrane-bound forms of cathepsin B display modified properties, e.g.resistance to inactivation at alkaline pH (23Sloane B.F. Rozhin J. Lah T.T. Day N.A. Buck M. Ryan R.E. Crissman J.D. Honn K.V. Adv. Exp. Med. Biol. 1988; 233: 259-268Crossref PubMed Scopus (16) Google Scholar). Previous results in the literature have shown that papain and cathepsin B are able to bind to laminin of basement membrane (24Dalet-Fumeron V. Boudjennah L. Pagano M. Arch. Biochem. Biophys. 1998; 358: 283-290Crossref PubMed Scopus (9) Google Scholar). These results are consistent with the proposed role of cysteine proteinases in degradation of extracellular matrix components. Therefore, the interaction of cysteine proteinases of the papain superfamily with glycosaminoglycans may be of significant interest for the understanding about the biological role of cysteine proteinases in tissue remodeling states. The bind of papain with glycosaminoglycans may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions, which can regulate its biological functions. In this study, we have investigated the influence of glycosaminoglycans, mainly heparin and heparan sulfate, upon papain activity. A combination of circular dichroism analysis, heparin-Sepharose affinity chromatography, and intramolecularly quenched fluorogenic substrate assays was used to characterize the interaction of papain with glycosaminoglycan. The papain was purchased from Calbiochem Co.; the concentration of the active enzyme was determined by titration using the cysteine proteinase inhibitor E-641 (25Ménard R. Khouri H.E. Plouffe C. Dupras R. Ripoll D. Vernet T. Tessier D.C. Laliberté F. Thomas D.Y. Storer A.C. Biochemistry. 1990; 29: 6706-6713Crossref PubMed Scopus (128) Google Scholar). Papain was stored at 4 °C in 50 mm sodium acetate buffer (pH 5.0) containing 10 μm methyl methane-thiosulfonate. The intramolecularly quenched fluorogenic peptide, Abz-AFRSSAQ-EDDnp, an analogue of Abz-AFRSAAQ-EDDnp (30Nagler D.K. Storer A.C. Portaro F.C.V. Carmona E. Juliano L. Ménard R. Biochemistry. 1997; 36: 12608-12615Crossref PubMed Scopus (151) Google Scholar), was synthesized using solid phase chemistry as described previously (26Hirata I.Y. Cezari M.H.S. Nakaie C.R. Boschcov P. Ito A.S. Juliano M. Juliano L. Lett. Peptide Sci. 1994; 1: 299-308Crossref Scopus (200) Google Scholar); the fluorogenic amidomethylcoumaryl substrate Cbz-FR-MCA and the papain irreversible inhibitor E-64 were purchased from Sigma. Heparin and heparan sulfate from bovine lung were a generous gift from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy); dermatan sulfate and chondroitin sulfate were purchased from Seikagaku Kogyo Co (Tokyo, Japan). Heparin-Sepharose resin was purchased from Amersham Pharmacia Biotech. The influence of glycosaminoglycans upon papain endopeptidase activity were determined spectrofluorometrically using the fluorogenic substrates Abz-AFRSSAQ-EDDnp and Cbz-FR-MCA. Fluorescence intensity was monitored on a thermostatic Hitachi F-2000 spectrofluorometer. The wavelengths were set at 380 nm for excitation and 440 nm for emission in the assays with the Cbz-FR-MCA substrate and 320 and 420 nm with Abz-AFRSSAQ-EDDnp. The enzyme was activated by incubation for 5 min at 37 °C in 50 mm sodium phosphate (pH 6.4) containing 200 mm NaCl, 1 mm EDTA, and 2 mm dithiothreitol. The measurements were done in the same buffer of papain activation, and the kinetic parameters were determined by measuring the initial rate of hydrolysis at various substrate concentrations in presence or absence of different glycosaminoglycan concentrations. The data obtained were analyzed by nonlinear regression using the program GraFit 3.01 (Erithacus Software Ltd.). The kinetic model depicted in Equation 1 can describe the effect of heparin on the hydrolysis of Abz-AFRSSAQ-EDDnp by papain,v=Vmax·[S]Ks1+[Hep]KH1+β·[Hep]α·KH+[S]1+[Hep]α·KH1+β·[Hep]α·KHEquation 1 where S is Abz-AFRSSAQ-EDDnp, Hep is heparin,K S is the substrate dissociation constant,K H is the apparent heparin dissociation constant, α is the parameter of K S perturbation, and β is the parameter of V max(k cat) perturbation. In the assays with the substrate Cbz-FR-MCA, the data obtained were also analyzed by nonlinear regression using GraFit 3.01. All progress curves obtained were exponential and could be best fitted according to the first-order relationship shown in Equation 2,P=P∞(1−e−Kobs′·t)Equation 2 where P and P ∞ are the product concentration at a given time and at infinite time, respectively, and K′obs is the observedk cat/K S rate Cbz-FR-MCA hydrolysis constant (27Tian W.X. Tsou C.L. Biochemistry. 1982; 21: 1028-1032Crossref PubMed Scopus (305) Google Scholar). The influence of heparin upon the observedk cat/K S rate constant can be described by Equation 3,Kobs′=Kobs(KH′+β·[Hep])KH′+[Hep]Equation 3 where K′obs andK obs are the observed rates in the presence and absence of heparin, respectively; K′H is the apparent heparin-papain dissociation constant at alkaline pH; Hep is heparin; and β is the parameter of limit forK obs in presence of heparin. For the determination of pH activity profiles, the kinetics of Cbz-FR-MCA hydrolysis was performed in absence or in presence of different heparin concentrations at 37 °C in 50 mm sodium phosphate, 50 mm citrate, or 50 mm borate containing 200 mm NaCl, 1 mm EDTA, and 2 mm dithiothreitol. The substrate concentration was kept well below the K m value. The progress of the reaction was monitored continuously by the fluorescence of the released product. The pH activity profiles were analyzed according to the model of by nonlinear regression as described previously (25Ménard R. Khouri H.E. Plouffe C. Dupras R. Ripoll D. Vernet T. Tessier D.C. Laliberté F. Thomas D.Y. Storer A.C. Biochemistry. 1990; 29: 6706-6713Crossref PubMed Scopus (128) Google Scholar). The kinetics of E-64 induced inactivation of papain were done under pseudo-first-order conditions (i.e. with an excess of inhibitor) at various heparin concentration in 50 mm sodium phosphate (pH 6.4) containing 200 mmNaCl, 1 mm EDTA, and 2 mm dithiothreitol. The reaction between papain and E-64 was monitored continuously in the presence of Cbz-FR-MCA. Progress of the reaction was monitored continuously by the fluorescence of the released product. All progress curves obtained were exponential decay and could be best fitted to the first-order relationship shown above (Equation 2). Enzyme and substrate were incubated under conditions similar to those described for the kinetic assays. The determination of cleaved bond for the peptide Abz-AFRSSAQ-EDDnp was proceeded on a reversed phase C-18 column using the Shimadzu SCL-8A HPLC system, equipped with a UV detector (220 nm) and a fluorescence detector with excitation and emission wavelengths set at 320 and 420 nm, respectively. The solvent system used was a 15 min gradient from 10 to 80% CH3CN, 0.1% trifluoroacetic acid at a flow rate of 1.7 ml/min. The products were collected, freeze-dried, and analyzed by mass spectrometry. Circular dichroism measurements in far ultraviolet regions (260 to 200 nm) of papain-glycosaminoglycan interactions were conducted in a JASCO J-700 spectropolarimeter scanning at rate of 10 nm/min at 25 °C. Cells of 0.05 cm for the far UV were used. The experiments were done in 50 mm sodium phosphate (pH 6.4) containing 200 mmNaCl, 0.07 mg/ml papain at various glycosaminoglycan concentrations. The observed ellipticity was normalized to units of degrees cm2/dmol. All dichroic spectra were smoothed and corrected by background subtraction for the spectrum obtained with buffer alone or buffer containing glycosaminoglycan. The spectra were analyzed for percent secondary structural elements by program based on comparison to the spectra obtained for the structures of known protein (28Sreerama N. Woody R.W. Anal. Biochem. 1994; 209: 32Crossref Scopus (946) Google Scholar). The influence of heparin in the papain helicity can be described by Equation 4,ΔHelix=Helixcontrol[Hep](β−1)KH+[Hep]Equation 4 where ΔHelix is the variation of papain helicity induced by heparin, K H is the apparent heparin-papain dissociation constant, Hep is heparin, and β is the parameter of limit for papain helicity induced by heparin. Papain (2.0 μg) dissolved in 50 mm sodium phosphate buffer (pH 6.4) was chromatographed on a heparin-Sepharose column (3 ml) and equilibrated in 50 mm sodium phosphate buffer (pH 6.4) at a flow rate of 0.5 ml/min. A linear NaCl gradient (0–2 m) was used to elute the bound material. The eluted fractions were monitored by papain enzymatic activity upon substrate Cbz-FR-MCA. The affinity of papain for heparin was evaluated by heparin-Sepharose chromatography. Papain was eluted from the heparin-Sepharose column at 1.0 m NaCl. This binding could be inhibited specifically by the previous addition of 100 μm of free heparin to papain solution (Fig.1). These data show that papain binding to heparin is mediated mainly by electrostatic interactions. These results led us to investigate the possible effect of heparin as well as other sulfated glycosaminoglycans, namely heparan sulfate, dermatan sulfate, and chondroitin sulfate, upon papain-catalyzed hydrolysis of the fluorogenic substrates Cbz-FR-MCA and Abz-AFRSSAQ-EDDnp. The effect of sulfated glycosaminoglycans on the papain endopeptidase activity was studied by monitoring the enzyme-catalyzed hydrolysis of the fluorogenic substrates. As observed, among the several sulfated polysaccharides studied only heparin and heparan sulfate exhibited patterns of interaction with papain, which varied according to the compound and substrate analyzed. The other sulfated glycosaminoglycans had no effect under the same experimental condition. Fig. 2 A shows that the presence of heparin in the papain kinetic assays results in a decrease in k cat values for the hydrolysis of Abz-AFRSSAQ-EDDnp. On the other hand, Fig. 2 B shows that heparin also markedly increases the affinity of the papain for the substrate Abz-AFRSSAQ-EDDnp. The effect of heparin upon papain endopeptidase activity can be described by a hyperbolic mixed type inhibition depicted in Equation 1. The efficiency of the system for the hydrolysis of the substrate can be altered by changing either theK S (parameter α) or V max(parameter β). The data were fitted to Equation 1 by using nonlinear regression, and the values for the constants were determined. The results show that heparin binds free papain (E) with a dissociation constant of K H = 27 ± 3 μm, and the complex enzyme-substrate (ES) with a dissociation constant of αK H = 3.5 ± 0.4 μm. Also, heparin induced a 9-fold increase in the affinity of papain for the substrate Abz-AFRSSAQ-EDDnp; the K S value was decreased from 0.66 ± 0.04 to 0.073 ± 0.006 μm in presence of heparin (α = 0.11 ± 0.01) (Fig. 1 B), whereas the k cat value in the presence of heparin was decreased 4-fold (β = 0.25 ± 0.01). However, the catalytic efficiency for this substrate in the presence of heparin was increased (β/α = 2.3 ± 0.2). Abz-AFRSSAQ-EDDnp is a very good substrate for papain, withk cat/K S = 3.0 107m−1·s−1; this substrate was chosen in order to position Phe and Arg residues inP 2 and P 1 and Ser residue in P′1. In this manner, the substrate covers S2, S1, and S′1, which are the main substrate binding sites in papain-like cysteine proteinases (29Turk D. Guncar G. Podobnik M. Turk B. Biol. Chem. Hoppe-Seyler. 1998; 379: 137-147Crossref PubMed Scopus (220) Google Scholar, 30Nagler D.K. Storer A.C. Portaro F.C.V. Carmona E. Juliano L. Ménard R. Biochemistry. 1997; 36: 12608-12615Crossref PubMed Scopus (151) Google Scholar, 31Barret A.J. Biochem. J. 1980; 187: 909-912Crossref PubMed Scopus (392) Google Scholar). The HPLC and mass spectrometry analysis showed that Arg-Ser is the only peptide bond cleaved by papain in this sequence. The presence of heparin did not change the pattern of cleavage of this peptide by papain. Curiously, heparin showed a different kinetic pattern upon papain when the preparation was assayed with the substrate Cbz-FR-MCA. Under these conditions, heparin showed a partial noncompetitive inhibition behavior. Fig. 3 shows the decrease in the observed first-order Cbz-FR-MCA hydrolysis rate by the presence of heparin. The observed k cat/K Srates of papain upon Cbz-FR-MCA in presence or absence of heparin were determined by using the kinetic model depicted in Equation 2. The kinetic model depicted in the Equation 3 can describe the effect of heparin on the observedk cat/K S. The data were fitted to Equation 3 by using nonlinear regression, and the values of the constants were obtained. The results show that heparin binds papain with a K′H of 4.0 ± 0.2 μm, and this interaction prevents the Cbz-FR-MCA hydrolysis. Heparin promoted a decrease of 5.5-fold in observed Cbz-FR-MCA hydrolysis second-order rate; in the absence of heparin, the second-order substrate hydrolysis rate was (4.53 ± 0.21) × 105m−1·s−1, and in the presence of heparin, the observed rate was (0.82 ± 0.08) × 105m−1·s−1(β = 0.19 ± 0.01). Most of this effect is related to the decrease in k cat; heparin-papain interaction did not affect Z-FR-MCA dissociation constant. The interaction of heparin with papain was also studied by verifying the heparin effect on E-64 papain inhibition activity. E-64 has been shown to bind in the S subsites of the papain and nucleophilic attack by Cys25 thiolate of enzyme occurs at the C3 atom of the epoxide. Also, the carboxyl-terminal group of E-64 forms an electrostatic interaction with the protonated His159imidazole ring (32Yamamoto D. Matsumoto K. Ohishi H. Ishida T. Inoue M. Kitamura K. Mizuno H. J. Biol. Chem. 1991; 266: 14771-14777Abstract Full Text PDF PubMed Google Scholar). Fig. 4 shows the decrease in the rate of E-64 inhibition by heparin. It was observed that the presence of 100 μm heparin decreases 5-fold the inhibitory activity of E-64 upon papain. TheK inac observed for E-64 was 0.36 ± 0.03 s−1, and in the presence of 100 μm heparin, the K inac was decreased to 0.073 ± 0.007 s−1, whereas the dissociation constant of E-64 (K i = 2.3 ± 0.2 μm) was not changed by the presence of heparin. Basically the same effect of heparin was obtained when 100 μm heparan sulfate was tested against the inhibitory activity of E-64. The effect of heparin upon papain conformation were examined by CD spectroscopy. Fig. 5 A shows that the addition of heparin to a solution containing 3 μm papain in 50 mm sodium phosphate buffer, pH 6.4, causes a significant change in the spectral envelope. In the presence of heparin, the ellipticity value at [θ]222 nmis decreased, showing that heparin increases the helicity of papain. Table I exhibits the secondary structure content of papain in the presence of different heparin concentrations. The fractions of the different papain structural types, α, β, and remainder (R), were computed from the CD spectra (200–260) shown in Fig. 5 A as described previously (28Sreerama N. Woody R.W. Anal. Biochem. 1994; 209: 32Crossref Scopus (946) Google Scholar). The data show a dramatic increase in the papain α-helix content induced by heparin, whereas β-structure and remainder content decreased. These changes are likely to be a reflex of the peptideheparin interaction. TableI also shows that the addition of 128 μm heparan sulfate increases papain α-helix content, very similar to the result obtained increasing the heparin concentration.Table ISecondary structure content of papain in the presence of different heparin concentrationsHeparinα-Helixβ-SheetRemainingμm%027.917.953.6 1633.017.050 3237.015.947.1 6439.514.745.8 9641.414.244.412842.214.043.820043.013.743.3128 μmheparan sulfate42.314.243.5 Open table in a new tab As expected, addition of 1 m NaCl to papain-heparin solution causes a spectral change consistent with the dissociation of the heparin-papain complex (Fig. 5 B). The spectrum obtained under high ionic strength conditions is essentially identical to the spectrum obtained for the papain alone in the presence of 1m NaCl. Fig. 6 shows that the increase on papain α-helices content induced by heparin is saturable, as predicted by Equation 4. The data show that the papain α-helices content was increased up to 67% in the presence of heparin, i.e.β = 1.67 ± 0.05. The value of the dissociation constant,K H = 33 ± 3 μm, measured by fitting the papain variation of helix content (ΔHelix) in function of heparin concentration is very similar to that obtained by analysis of substrate hydrolysis, K H = 27 ± 3 μm. In order to probe the polysaccharide sequence requirements for papain interaction, the effects of other polysulfated polysaccharides were tested. Table II shows that, besides heparin, only heparan sulfate was able to decrease the substrateK S value, and it simultaneously induced α-helix content in papain. On the other hand, dermatan sulfate and chondroitin sulfate, at same heparin molar concentration, were not able to decrease the K S value and did not induce α-helix structure in papain.Table IIα-Helix content and KS values of papain in the presence of different glycosaminoglycansGAGKSα-Helixμm%Control0.7427.9Heparin (32 μm)0.1237.0Heparan sulfate (32 μm)0.1635.4Dermatan sulfate (32 μm)0.7228.0Chondroitin sulfate (32 μm)0.7327.5 Open table in a new tab In general, the papain activity is related to the presence of a thiolate-imidazolium ion pair between the active site Cys25and His159. The catalytic residue Cys25 is located in the central α-helix at papain L-domain, and His159 is located at the R-domain. The active site (Cys25 and His159) is situated at interdomain interface forming a V-shaped cleft situated on the top of the papain (33Polgar L. Mechanism of Protease Action. CRC Press Inc., Boca Raton, FL1989Google Scholar, 34Kamphuis I.G. Kalk K.H. Swarte M.B.A. Drenth J. J. Mol. Biol. 1984; 179: 233-257Crossref PubMed Scopus (466) Google Scholar). Hydrogen bonding and electrostatic and interdomain hydrophobic interactions stabilize the papain active site. The deprotonation of His159, catalyzed by OH−ions, is considered a crucial event for alkaline pH-induced inactivation of cysteine proteinases. This process is thought to be reversible for papain and irreversible for cathepsin B and L (35Dufour E. Dive V. Toma F. Biochim. Biophys. Acta. 1988; 995: 58-64Crossref Scopus (20) Google Scholar, 36Turk B. Dolenc I. Zerovnic E. Turk D. Gubensek F. Turk V. Biochemistry. 1994; 33: 14800-14806Crossref PubMed Scopus (73) Google Scholar, 37Turk B. Dolenc I. Turk V. Bieth J.G. Biochemistry. 1993; 32: 375-380Crossref PubMed Scopus (130) Google Scholar). Table III shows the influence of heparin upon pH activity profile of the hydrolysis of Cbz-FR-MCA by papain. It is very interesting to note that the pH activity profile for the hydrolysis of Cbz-FR-MCA by papain in the presence of heparin is shifted to the right, the value of the pK 1obs was shifted from 4.54 to 5.03, and the pK 2obs was increased from 8.45 to 8.93. These results suggest that the presence of heparin is decreasing the rate of papain His159 imidazolium deprotonation, increasing the activity of the papain by preserving its thiolate-imidazolium ion pair at alkaline pH.Table IIIKinetic parameters for hydrolysis of Cbz-FR-MCA by papain in the presence of heparinkcatKSpHoptpK1obspK2obss−1μmControlaRef. 40.41.6 ± 6.889 ± 66.54.54 ± 0.298.45 ± 0.05Papain + 100 μmheparin8.2 ± 0.693 ± 57.05.03 ± 0.048.93 ± 0.05a Ref. 40Vernet T. Tessier D.C. Chatellier J. Plouffe C. Lee T.S. Thomas D.Y. Storer A.C. Ménard R. J. Biol. Chem. 1995; 270: 16645-16652Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar. Open table in a new tab Table IV shows that a drastic decrease in the papain α-helix content was observed when the enzyme was exposed at pH 7.4, whereas the β-sheet content increased. At pH 6.4, the α-helix and β-sheet contents were 28 and 18%, respectively, and at pH 7.4, the α-helix and β-sheet contents were 18.5 and 27.4%, respectively. However, when papain was preincubated with heparin, the amount of α-helix structure disruption, induced by alkaline pH, was decreased. These data corroborate the results shown in Table III, suggesting that the thiolate-imidazolium ion pair, at alkalyne pH, is preserved by the papain-heparin interaction, stabilizing the papain α-helix structures.Table IVFar UV (200–260 nm) CD spectra of papain at different pH levelsConditionsα-Helixβ-SheetRemainingTotal%pH 6.427.917.953.699.4pH 7.418.527.454.1100.0pH 7.4 + 100 μmheparin24.822.253.5100.5R-X analysis (34)28.014.058.0100.0The CD analysis of papain proceeded at pH 6.4 and 7.4 and at pH 7.4 in the presence of 100 μm heparin as described under "Experimental Procedures." The Sreerama and Woody analysis (28Sreerama N. Woody R.W. Anal. Biochem. 1994; 209: 32Crossref Scopus (946) Google Scholar) of the papain spectra is given below. Open table in a new tab The CD analysis of papain proceeded at pH 6.4 and 7.4 and at pH 7.4 in the presence of 100 μm heparin as described under "Experimental Procedures." The Sreerama and Woody analysis (28Sreerama N. Woody R.W. Anal. Biochem. 1994; 209: 32Crossref Scopus (946) Google Scholar) of the papain spectra is given below. Several reports in the literature show that papain can be purified using cation-exchange chromatography (20Barret A.J. Kirschke H. Methods Enzymol. 1981; 80: 535-561Crossref PubMed Scopus (1729) Google Scholar, 25Ménard R. Khouri H.E. Plouffe C. Dupras R. Ripoll D. Vernet T. Tessier D.C. Laliberté F. Thomas D.Y. Storer A.C. Biochemistry. 1990; 29: 6706-6713Crossref PubMed Scopus (128) Google Scholar, 38Solı́s-Mendiola S. Arroyo-Reyna A. Hernández-Arana A. Biochim. Biophys. Acta. 1992; 1118: 288-292Crossref PubMed Scopus (21) Google Scholar); these results suggest that papain can bind anionic polysaccharides, such as heparin and heparan sulfate. The affinity of papain for heparin was evaluated by heparin-Sepharose chromatography. We observed that papain possessed high affinity binding to heparin, being eluted at 1.0 mNaCl from a heparin-Sepharose column. This interaction is specific, because this binding was disrupted by the previous addition of 100 μm of free heparin to papain solution (Fig. 1). These data show that papain binding to heparin is mediated mainly by electrostatic interactions. The binding of heparin to the papain perturbs its catalytic activity upon fluorogenic substrates. It was observed that heparin inhibits papain endopeptidase activity upon the substrate Abz-AFRSSAQ-EDDnp by a hyperbolic mixed type inhibition fashion (Fig. 2). The presence of heparin results in a decrease in k cat values (β = 0.25 ± 0.01) but also markedly increases the affinity of the enzyme for the substrate (α = 0.11 ± 0.01),i.e. α < 1, β < 1, and β > α. Likewise, at high substrate concentration, the affinity of papain for heparin is increased. It was observed that heparin binds free papain (E) with a dissociation constant of K H = 27 ± 3 μm, and heparin binds the complex enzyme substrate (ES) with a dissociation constant of αK H = 3.5 ± 0.4 μm. On the other hand, heparin only perturbed thek cat value for the substrate Cbz-FR-MCA that was decreased 5-fold, β = 0.20 ± 0.01, whereas theK S value for this substrate was not changed in the presence of heparin (Fig. 3). Also, heparin only prevents papain E-64 inhibition by decreasing the rate of inactivation (Fig. 4). TheK inac (inactivation constant) observed for E-64 was 0,36 ± 0.03 s−1, and in presence of 100 μm of heparin, the K inac was decreased to 0.073 ± 0.007 s−1, whereas the dissociation constant of E-64 (K i = 2.3 ± 0.2 μm) was not changed by the presence of heparin. It is well known that E-64 interacts with papain mainly in theS 1 and S 2 subsite (32Yamamoto D. Matsumoto K. Ohishi H. Ishida T. Inoue M. Kitamura K. Mizuno H. J. Biol. Chem. 1991; 266: 14771-14777Abstract Full Text PDF PubMed Google Scholar). Also, the substrate Cbz-FR-MCA covers the papain subsites at theS 1 and S 2 positions, whereas the substrate Abz-AFRSSAQ-EDDnp covers from theS 3 to the S′4 position. Taken together, these results suggest that heparin is modulating the dissociation constant for the substrate Abz-AFRSSAQ-EDDnp by perturbing the papain in S′n positions. The decrease promoted by heparin in k cat values of the papain for the substrates Abz-AFRSSAQ-EDDnp and Cbz-FR-MCA is similar to the effect promoted by heparin in the papain E-64 inactivation rate. These results suggest that heparin binding is perturbing the papain active site in a similar manner. Binding to heparin significantly increases the α-helix content of the papain, and the binding event can be monitored by CD analysis. This binding is marked by significant changes in the shape and position of the CD spectral envelope (Fig. 5 A). As expected, according to the data obtained from the heparin-Sepharose experiments, the interaction between papain and heparin was abolished by the addition of 1 m NaCl (Fig. 5 B). The increase of the papain α-helix content is a reflex of the papain-heparin interaction, because the dissociation constant, K H = 33 ± 3 μm, measured by fitting the papain α-helix content variation in the function of heparin concentration (Fig. 6), is very similar to that obtained by analysis of substrate hydrolysis,K H = 27 ± 3 μm. These results strongly suggest that the conformational change induced by heparin leads to an increase in the affinity of the papain for the substrate Abz-AFRSSAQ-EDDnp. Table I shows that heparin increases the helical content of the papain by increasing the number of residues in the helical conformation; it seems to be related to the decrease of the β-sheet and remaining structures content. The data obtained also show that heparan sulfate is able to cause a spectral change in papain very similar to those obtained by increasing the heparin concentration. Table II shows that the interaction between heparin or heparan sulfate and papain is quite specific, because other sulfated glycosaminoglycans, namely dermatan sulfate and chondroitin sulfate, were not able to increase the papain affinity for the substrate or induce α-helix structures in the papain. The alkaline pH-induced inactivation, as well as the unfolding of human cathepsin B (36Turk B. Dolenc I. Zerovnic E. Turk D. Gubensek F. Turk V. Biochemistry. 1994; 33: 14800-14806Crossref PubMed Scopus (73) Google Scholar) and cathepsin L (35Dufour E. Dive V. Toma F. Biochim. Biophys. Acta. 1988; 995: 58-64Crossref Scopus (20) Google Scholar, 37Turk B. Dolenc I. Turk V. Bieth J.G. Biochemistry. 1993; 32: 375-380Crossref PubMed Scopus (130) Google Scholar), was shown to be a first-order process, indicating that this inactivation of cysteine proteinases correlates with protein stability. The deprotonation of His159, catalyzed by OH− ions, is considered a crucial event for alkaline pH-induced inactivation of cysteine proteinases. The break of the thiolate-imidazoliun ion pair influences ionization and solvent exposure of some charged residues located at the interdomain interface, resulting in conformational changes that promote destabilization of the central α-helix (35Dufour E. Dive V. Toma F. Biochim. Biophys. Acta. 1988; 995: 58-64Crossref Scopus (20) Google Scholar, 36Turk B. Dolenc I. Zerovnic E. Turk D. Gubensek F. Turk V. Biochemistry. 1994; 33: 14800-14806Crossref PubMed Scopus (73) Google Scholar, 37Turk B. Dolenc I. Turk V. Bieth J.G. Biochemistry. 1993; 32: 375-380Crossref PubMed Scopus (130) Google Scholar). Table III shows that heparin shifts the papain pH activity profile to the right, allowing papain to be active at alkaline pH. In addition, according to the data presented in Table IV, the presence of heparin reduces the loss of papain α-helix content induced by alkaline pH. Heparin increases the stability of papain at alkaline pH, which is a reflex of the higher α-helix amount observed for the papain-heparin complex compared with that for the papain alone. Although a heparin-binding domain in papain has not yet been demonstrated, the papain sequence 188–191 (RIKR) is a putative heparin-binding site, as previously suggested (39Cardin A.D. Weintraub H.J. Artheriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). Cardin and Weintraub (39Cardin A.D. Weintraub H.J. Artheriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) proposed that the consensus heparin-binding sequences might occur in either helices or β-strand structures. The putative heparin-binding site in the 188–191 stretch of the papain displays β-strand structure (34Kamphuis I.G. Kalk K.H. Swarte M.B.A. Drenth J. J. Mol. Biol. 1984; 179: 233-257Crossref PubMed Scopus (466) Google Scholar). This cationic sequence is located adjacent to the residues Asn175 and Trp177. In papain, it is known that Asn175 (40Vernet T. Tessier D.C. Chatellier J. Plouffe C. Lee T.S. Thomas D.Y. Storer A.C. Ménard R. J. Biol. Chem. 1995; 270: 16645-16652Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) and Trp177 (41Baker E.N. Drenth J. Biological Macromolecules and Assemblies. John Wiley & Sons, New York1987Google Scholar) contribute to the electrostatic field, and the residues influence the formation of the catalytically active thiolate-imidazolium ion pair, whereas residue Trp177 is also involved in enzyme-substrate interactions (41Baker E.N. Drenth J. Biological Macromolecules and Assemblies. John Wiley & Sons, New York1987Google Scholar). Binding of heparin or heparan sulfate to papain may change the relative orientation of the surface structures, forcing a conformational change in the protein. This conformational change could be communicated to the rest of the protein via tertiary structure or disulfide bonds (42Lellouch A., C. Lansbury Jr., P.T. Biochemistry. 1992; 31: 2279-2285Crossref PubMed Scopus (19) Google Scholar). Both Arg and Lys residues are found in the established heparin-binding domains of various proteins. Also, the heparin binding may stabilize the papain helices by eliminating detrimental electrostatic interactions, and the negatively charged sulfate and carboxylate groups of heparin may neutralize positive charges that might otherwise contribute to helix destabilization (43Ferran D.S. Sobel M. Harris R.B. Biochemistry. 1992; 31: 5010-5016Crossref PubMed Scopus (46) Google Scholar). In conclusion, the present results show that the conformational change induced by heparin binding in papain is specific; this change leads to an increase in the affinity of the papain to the substrate Abz-AFRSSAQ-EDDnp and stabilizes the central α-helix in the active site, preserving its functional structure even at alkaline pH. Recently, we have made similar observations by studying human cathepsin B in the presence of heparin and heparan sulfate, suggesting that the bind of papain with glycosaminoglycans is representative of other mammalian cysteine proteinase-glycosaminoglycan interactions. Heparin and heparan sulfate was also able to binding human cathepsin B, and this interaction protects the human cathepsin B against alkaline pH-induced inactivation. 2P. C. Almeida, I. L. Nantes, C. C. A. Rizzi, W. A. S. Júdice, J. R. Chagas, L. Juliano, H. B. Nader, and I. L. S. Tersariol, manuscript in preparation. Our results show that heparan sulfate may be an important binding site of cysteine proteinases at basement membranes; this binding stabilizes these enzymes at pH 7.4. Moreover, the binding of cysteine proteinases with basement membranes is of significant interest for understanding the biological role of cysteine proteinases in tumor invasion and other types of tissue remodeling states. We thank Drs. Michel Goldberg (Institute Pasteur, Paris, France) and Robert Ménard (Biotechnology Research Institute, Montreal, Quebec, Canada) for helpful discussions and Dr. Adelaide Faljoni-Alário (Instituto de Química-Universidade de São Paulo, São Paulo, Brazil) for helping in the CD analysis.
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