J Biol Chem, Vol. 274, Issue 43, 30433-30438, October 22, 1999

Cysteine Proteinase Activity Regulation
A POSSIBLE ROLE OF HEPARIN AND HEPARIN-LIKE GLYCOSAMINOGLYCANS^*

Paulo C. Almeida, Iseli L. Nantes, Cláudia C. A. Rizzi, Wagner A. S. Júdice^§, Jair R. Chagas^§, Luiz Juliano^§, Helena B. Nader^¶, and Ivarne L. S. Tersariol

From the  Centro Interdisciplinar de Investigação Bioquímica, Universidade Mogi das Cruzes, Prédio II, (Centro de Ciências Biomédicas), sala 22.09, Av. Dr. Candido X. de Almeida Souza 200, CP 411, CEP 08780-911, Mogi das Cruzes, SP, Brazil, and the ^§ Departamento de Biofísica and the ^¶ Disciplina de Biologia Molecular, Instituto Nacional de Farmacologia, Universidade Federal de São Paulo/Escola Paulista de Medicina, Rua 3 de maio 100, CEP 04044-020, São Paulo, SP, Brazil

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -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 -helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce -helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1-3). Most cellular heparan sulfate, at the cell surface and in extracellular matrix, derives from the syndecan proteoglycan family (4). 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 (5).

It has been shown that heparin can modify the activities of some serine proteinases and its natural inhibitors in vitro (6-8) and also that heparan sulfate proteoglycans, syndecan-1 ectodomain, and syndecan-4 ectodomain are shed into acute inflammatory wound fluids (9). The purified syndecan-1 ectodomain protects cathepsin G from inhibition by 1-antichymotrypsin and squamous cell carcinoma antigen 2, and it protects elastase from inhibition by 1-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 (10). 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 (9, 10).

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 (11). 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 (12, 13), parasite infection (14, 15), arthritis (16), and other types of inflammatory injury (17). Cathepsins B and L can participate in metastasis formation by degradation of several extracellular matrix components (18-20).

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 (21). Confocal microscopy image analysis indicated that cathepsin B was associated with the external basal cell surface in the murine B16 amelanotic melanoma cells (22). It has been shown that membrane-bound forms of cathepsin B display modified properties, e.g. resistance to inactivation at alkaline pH (23). Previous results in the literature have shown that papain and cathepsin B are able to bind to laminin of basement membrane (24). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The papain was purchased from Calbiochem Co.; the concentration of the active enzyme was determined by titration using the cysteine proteinase inhibitor E-64¹ (25). 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 (30), was synthesized using solid phase chemistry as described previously (26); 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.

Enzyme Assays-- 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,

(Eq. 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,  is the parameter of K_S perturbation, and  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,

(Eq. 2)

where P and P are the product concentration at a given time and at infinite time, respectively, and K'_obs is the observed k_cat/K_S rate Cbz-FR-MCA hydrolysis constant (27). The influence of heparin upon the observed k_cat/K_S rate constant can be described by Equation 3,

(Eq. 3)

where K'_obs and K_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  is the parameter of limit for K_obs in presence of heparin.

The Influence of Heparin upon Papain Activity at Different pH Levels-- 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 (25).

Effect of Heparin on E-64 Induced Inactivation of Papain-- 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 mM NaCl, 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).

Identification of the Cleavage Site for Abz-AFRSSAQ-EDDnp Substrate-- 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 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 mM NaCl, 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 (28). The influence of heparin in the papain helicity can be described by Equation 4,

(Eq. 4)

where Helix is the variation of papain helicity induced by heparin, K_H is the apparent heparin-papain dissociation constant, Hep is heparin, and  is the parameter of limit for papain helicity induced by heparin.

Heparin-Sepharose Affinity Chromatography-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Papain Binds Heparin-Sepharose Column-- 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.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Papain binds heparin-Sepharose column. Papain (2.0 µg) dissolved in 50 mM sodium phosphate buffer (pH 6.4) was chromatographed on a heparin-Sepharose column (). Papain was preincubated with 100 µM heparin and then submitted to the heparin-Sepharose column ().

Effect of Heparin and Heparan Sulfate upon the Papain Endopeptidase Activity-- 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. 2A 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. 2B 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 the K_S (parameter ) or V_max (parameter ). 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 K_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 ( = 0.11 ± 0.01) (Fig. 1B), whereas the k_cat value in the presence of heparin was decreased 4-fold ( = 0.25 ± 0.01). However, the catalytic efficiency for this substrate in the presence of heparin was increased (/ = 2.3 ± 0.2). Abz-AFRSSAQ-EDDnp is a very good substrate for papain, with k_cat/K_S = 3.0 10⁷ M¹·s¹; this substrate was chosen in order to position Phe and Arg residues in P₂ and P₁ 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 (29-31). 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.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effect of heparin on Abz-AFRSSAQ-EDDnp hydrolysis by papain. The influence of heparin concentration upon papain endopeptidase activity was determined spectrofluorometrically as described under "Experimental Procedures." Heparin promotes a decrease in papain kcat values (A) and also increases the affinity of papain for the substrate (B)

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_S rates 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 observed k_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) × 10⁵ M¹·s¹, and in the presence of heparin, the observed rate was (0.82 ± 0.08) × 10⁵ M¹·s¹ ( = 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.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of heparin on Cbz-FR-MCA hydrolysis by papain. The influence of heparin concentration upon papain activity was determined spectrofluorometrically as described under "Experimental Procedures."

Heparin Prevents the Inhibitory Activity of E-64 upon Papain-- 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 His¹⁵⁹ imidazole ring (32). 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. The K_inac observed for E-64 was 0.36 ± 0.03 s¹, and in the presence of 100 µM heparin, the K_inac was decreased to 0.073 ± 0.007 s¹, 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.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Heparin prevents the inhibitory activity of E-64 upon papain. Papain activity was determinated in the presence of different concentrations of E-64 (0.35-2.5 µM) in the absence () and in the presence () of 100 µM heparin.

Effects of Heparin on Papain Circular Dichroism Spectra-- The effect of heparin upon papain conformation were examined by CD spectroscopy. Fig. 5A 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 []_{222 nm} is 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, , , and remainder (R), were computed from the CD spectra (200-260) shown in Fig. 5A as described previously (28). The data show a dramatic increase in the papain -helix content induced by heparin, whereas -structure and remainder content decreased. These changes are likely to be a reflex of the peptideheparin interaction. Table I also shows that the addition of 128 µM heparan sulfate increases papain -helix content, very similar to the result obtained increasing the heparin concentration.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects of heparin on papain circular dichroism spectra. A, papain CD spectra were determined at pH 6.4 as described under "Experimental Procedures." The CD spectra were obtained in the absence of heparin () and in the presence of 32 (- - - - -) and 64 (· · · · ·) µM heparin. B, the papain CD spectra were obtained at 1.0 M NaCl in the absence () and in the presence (- - - - -) of 64 µM heparin.

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. 5B). The spectrum obtained under high ionic strength conditions is essentially identical to the spectrum obtained for the papain alone in the presence of 1 M NaCl.

Fig. 6 shows that the increase on papain -helices content induced by heparin is saturable, as predicted by Equation 4. The data show that the papain -helices content was increased up to 67% in the presence of heparin, i.e.  = 1.67 ± 0.05. The value of the dissociation constant, K_H = 33 ± 3 µM, measured by fitting the papain variation of helix content (Helix) in function of heparin concentration is very similar to that obtained by analysis of substrate hydrolysis, K_H = 27 ± 3 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   The influence of heparin in the papain helicity. The amount of papain -helix content induced by heparin was verified as described under "Experimental Procedures."

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 substrate K_S value, and it simultaneously induced -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 -helix structure in papain.

View this table: [in this window] [in a new window]   Table II -Helix content and KS values of papain in the presence of different glycosaminoglycans

The Influence of Heparin upon Papain pH Activity Profile-- In general, the papain activity is related to the presence of a thiolate-imidazolium ion pair between the active site Cys²⁵ and His¹⁵⁹. The catalytic residue Cys²⁵ is located in the central -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 (33, 34). 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 (35-37).

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 pK₁^obs was shifted from 4.54 to 5.03, and the pK₂^obs 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 IV shows that a drastic decrease in the papain -helix content was observed when the enzyme was exposed at pH 7.4, whereas the -sheet content increased. At pH 6.4, the -helix and -sheet contents were 28 and 18%, respectively, and at pH 7.4, the -helix and -sheet contents were 18.5 and 27.4%, respectively. However, when papain was preincubated with heparin, the amount of -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 -helix structures.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several reports in the literature show that papain can be purified using cation-exchange chromatography (20, 25, 38); 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 M NaCl 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 ( = 0.25 ± 0.01) but also markedly increases the affinity of the enzyme for the substrate ( = 0.11 ± 0.01), i.e.  < 1,  < 1, and  > . 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 K_H = 3.5 ± 0.4 µM. On the other hand, heparin only perturbed the k_cat value for the substrate Cbz-FR-MCA that was decreased 5-fold,  = 0.20 ± 0.01, whereas the K_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). The K_inac (inactivation constant) observed for E-64 was 0,36 ± 0.03 s¹, and in presence of 100 µM of heparin, the K_inac was decreased to 0.073 ± 0.007 s¹, 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 the S₁ and S₂ subsite (32). Also, the substrate Cbz-FR-MCA covers the papain subsites at the S₁ and S₂ positions, whereas the substrate Abz-AFRSSAQ-EDDnp covers from the S₃ 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 -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. 5A). 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. 5B). The increase of the papain -helix content is a reflex of the papain-heparin interaction, because the dissociation constant, K_H = 33 ± 3 µM, measured by fitting the papain -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 -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 -helix structures in the papain.

The alkaline pH-induced inactivation, as well as the unfolding of human cathepsin B (36) and cathepsin L (35, 37), 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 -helix (35-37).

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 -helix content induced by alkaline pH. Heparin increases the stability of papain at alkaline pH, which is a reflex of the higher -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 (39). Cardin and Weintraub (39) proposed that the consensus heparin-binding sequences might occur in either helices or -strand structures. The putative heparin-binding site in the 188-191 stretch of the papain displays -strand structure (34). This cationic sequence is located adjacent to the residues Asn¹⁷⁵ and Trp¹⁷⁷. In papain, it is known that Asn¹⁷⁵ (40) and Trp¹⁷⁷ (41) 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 (41).

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 (42). 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 (43).

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 -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.² 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 97/13133-4 and Fundação de Amparo ao Ensino e Pesquisa-Universidade de Mogi das Cruzes, Brazil.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 55-11-4798-7102; Fax: 55-11-4798-7288; E-mail: pcezar@mandic.com.br.

² P. 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.

	ABBREVIATIONS

The abbreviations used are: E-64, 1-[[(L-trans-epoxysuccinyl)-L-leucyl]amino]-4-guanidino-butane; Cbz-FR-MCA, carbobenzoxyl-L-phenylalanyl-L-arginine-4-methylcoumarinyl-7-amide; Abz-AFRSSAQ-EDDnp, ortho-aminobenzoyl-L-alanyl-L-phenylalanyl-L-arginyl-L-seryl-L-seryl-L-alanyl-L-glutamine N-(ethylenediamine)-2,4-dinitrophenylamide; HPLC, high pressure liquid chromatography.

	REFERENCES

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