Cathepsin B Activity Regulation

HEPARIN-LIKE GLYCOSAMINOGLYCANS PROTECT HUMAN CATHEPSIN B FROM ALKALINE pH-INDUCED INACTIVATION*

Paulo C. AlmeidaDagger , Iseli L. NantesDagger , Jair R. ChagasDagger , Cláudia C. A. RizziDagger , Adelaide Faljoni-Alario§, Euridice Carmona, Luiz Juliano||, Helena B. Nader**, and Ivarne L. S. TersariolDagger DaggerDagger

From the Dagger  Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Prédio I, Centro de Ciências Tecnológicas, sala 1S-15, Av. Dr. Candido X. de Almeida Souza 200, CEP 08780-911, Mogi das Cruzes, SP, Brazil, § Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil,  Laboratório de Farmacologia, Instituto Butantã, São Paulo, SP, Brazil, and || Departamento de Biofísica and the ** Disciplina de Biologia Molecular, Universidade Federal de São Paulo/Escola Paulista de Medicina, Instituto Nacional de Farmacologia, São Paulo, SP, Brazil

Received for publication, May 4, 2000, and in revised form, September 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its alpha -helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Júdice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B alpha -helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t1/2) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His111 residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heparan sulfate is an ubiquitous glycosaminoglycan of animal cells (1). These classes of compounds are heteropolysaccharides made up of repeating units of disaccharides, an uronic acid residue, either D-glucuronic acid or L-iduronic acid, and D-glucosamine with N- and 6-O-sulfates and N-acetyl substitutions (2). Heparan sulfate occurs at the cell surface and in extracellular matrix as proteoglycans. Most of cellular heparan sulfate derives from the syndecans and glypicans proteoglycans. The syndecan family are associated with the cell membranes via transmembrane core proteins (3, 4), and the glypican family is anchored by glycosilyl phosphatidylinositol-anchor core proteins (5). Also, heparan sulfate proteoglycans are present in basement membranes performing the perlecan family (6).

Heparan sulfate and heparin are particular among glycosaminoglycans in their ability to bind a large number of different proteins. Heparin-like glycosaminoglycans play a complex role in the extracellular matrix, regulating a wide variety of biological process, including hemostasis, inflammation, angiogenesis, growth factors, cell adhesion, and others (7). Proteolytic enzymes control many of these biological process. Several reports in the literature have demonstrated that heparin-like glycosaminoglycans can modulate the activity of some serine proteinases and their natural inhibitors (8-11). On the other hand, the interaction of cysteine proteinases with glycosaminoglycans has not been completely explored. In mammalians, lysosomal cysteine proteinases have been implicated in a variety of diseases involving tissue remodeling states, such as inflammation (12), parasite infection (13), and tumor metastasis (14). Cathepsin B shows close structural homology to the other cysteine proteases of the papain family (15). The main feature that distinguishes cathepsin B is the presence of a large insertion loop structure, termed occluding loop, which covers the active site, occupying the S2'- S3' subsites of the enzyme (16).

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, can participate in tumor invasion by degradation of extracellular matrix components (17). This can take place either intracellularly by heterophagosomal activity of tumors cell (18) or extracellularly by cell surface associated cathepsin B (14). It has been demonstrated that the presence of cathepsin B at plasma membrane results in focal dissolution of extracellular matrix proteins and enables the tumor cell to invade (19, 20). Trafficking and targeting of lysosomal enzymes is mostly mediated by mannose-6-phosphate receptor pathways (21). However, several reports show that this class of receptors is not sufficient for targeting of lysosomal enzymes along intracellular routes, either by an alteration in these receptors (22) or by changes in glycosylation pattern of lysosomal enzymes as observed for cathepsin B in carcinoma cells (23). Indeed, mannose-6-phosphate-independent targeting has been proposed for cathepsin B in normal cell (24) and human colon carcinoma cell lines (25). A high level of cathepsin B and qualitative changes in cathepsin B protein expression, including abnormal pattern of glycosylation, may be important in maintaining the malignant phenotype in carcinoma cell (23). Alterations in cathepsin B expression, processing, and cellular localization have been observed in several human tumor tissue; clinical investigations have shown that cathepsin B are highly predictive indicator for prognosis and diagnosis in cancer (26).

The mechanism of secretion and insertion of cathepsin B at the plasma membrane are not fully understood (27). Cathepsin B is secreted by normal and by tumor cells as the precursor forms (28), whereas many types of tumors cells may also release mature, active form of cathepsin B (29). However, it is not know whether the precursors are activated at the plasma membrane or extracellularly. It has been shown that the activation of cathepsins B and L occur autocatalytically triggered by acidic pH and also by anionic polysaccharides such as dextran sulfate and heparin (30, 31). The mature form of cathepsin B and L have been shown to be rapidly inactivated at neutral or alkaline pH end by its endogenous proteins inhibitors, mainly from the cystatin family (15). On the other hand, it has been shown that membrane-bound forms of cathepsin B are very resistant to inactivation at neutral pH (32).

Recently, we have shown that heparin and heparan sulfate bind papain specifically; this interaction induces an increase of alpha -helix content of papain, which stabilizes the enzyme structure even at alkaline pH (33). These results strongly suggest that heparan sulfate may be an important binding site of cysteine proteinases at cell surface and basement membrane. Therefore, the study of the interaction of cathepsin B with glycosaminoglycans is of significant interest for the understanding about the biological role of this enzyme. In this work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of cathepsin B with glycosaminoglycans.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cathepsin B from human liver was purchased from Calbiochem Co.; nonglycosylated cathepsin B S115A, denominated wild-type cathepsin, and its punctual mutants H110A and H111A were subcloned, as proenzymes, into the vector pPIC9 and expressed in yeast Pichia pastoris as described previously (34). The concentration of the active enzyme was determined by titration using the cysteine proteinase inhibitor E-641 (35). Active forms of cathepsin B were stored at 4 °C in 50 mM sodium acetate buffer (pH 5.0) containing 10 µM methyl methane-thiosulfonate. The irreversible inhibitor E-64, azocasein, the fluorogenic amidomethylcoumaryl substrate Cbz-FR-MCA, and dextran sulfate (5,000 Da) were purchased from Sigma. The intramolecularly quenched fluorogenic substrate Abz-FRA(epsilon -Dnp)K was synthesized using solid phase chemistry as described previously (36). In the experiments we have used a size-defined (10 kDa) bovine lung heparin (The Upjohn Co.), prepared by using size exclusion column approach (2, 7); heparan sulfate (16,000 Da) from bovine lung were a generous gift from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy) (2); dermatan sulfate (12,000 Da) and chondroitin sulfate (25,000 Da) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Heparin-Sepharose resin was purchased from Amersham Pharmacia Biotech.

Kinetic Measurements-- Cathepsin B activities were monitored spectrofluorometrically using the fluorogenic substrates Cbz-FR-MCA and Abz-FRA(epsilon -Dnp)K on a thermostatic Hitachi F-2000 spectrofluorometer. For Cbz-FR-MCA assays, the excitation and emission wavelengths were set at 380 and 460 nm, respectively. The assay with Abz-FRA(epsilon -Dnp)K was monitored at 420 nm using an excitation wavelength of 320 nm (37). Prior to the assay, the methyl methane-thiosulfonate-inhibited enzymes were activated by incubation for 5 min at 25 °C in 50 mM sodium phosphate (pH 6.0), 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol. For the determination of pH activity profiles, the kinetics of Cbz-FR-MCA and Abz-FRA(epsilon -Dnp)K hydrolysis were performed in absence or in presence of different glycosaminoglycans concentrations at 37 °C in 50 mM sodium phosphate (pH 5.8-8.0), 50 mM citrate (pH 3.0-5.9) containing 200 mM NaCl, 1 mM EDTA, and 2 mM DTT. The substrate concentrations were kept 20-fold below the Km values. The progress of the reaction was continuously monitored by the fluorescence of the released product. The initial rates were determined, and the kcat/KM values were obtained by dividing the initial rates by enzyme and substrate concentrations. The pH activity profiles for cathepsin B are extremely complex, which precludes the use of well defined models to curve fit the data. Therefore, the empirical equations were included solely for clarity of the graphical display (34). The kinetic model depicted in Equation 1 can describe the effect of heparin on the hydrolysis of Abz-FRA(epsilon -Dnp)K by cathepsin B.


v=<FR><NU>v<SUB><UP>max</UP></SUB> · [<UP>S</UP>]</NU><DE>K<SUB>s</SUB><FENCE><UP>I</UP>+<FR><NU>[<UP>I</UP>]</NU><DE>K<SUB>I</SUB></DE></FR></FENCE>+[<UP>S</UP>]</DE></FR> (Eq. 1)
where S is Abz-FRA(epsilon -Dnp)K, I is heparin, KS is the substrate dissociation constant, and KI is heparin dissociation constant. The influence of heparin upon proteolytic activity of cathepsin B was also assayed against protein substrate using azocasein as described previously (38).

Effect of Heparin on E-64 Induced Inactivation of Cathepsin B-- The kinetics of cathepsin B inactivation by E-64 was done under pseudo first-order conditions at various heparin concentration as described previously (33).

The Alkaline pH-induced Inactivation-- The kinetics of alkaline pH-induced inactivation of cathepsins B and its mutants were done at different glycosaminoglycan concentrations in 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol. The inactivation of the enzymes was performed in the presence of 10 µM Cbz-FR-MCA. Progress of the reaction was monitored continuously by the fluorescence of the released product. The obtained exponential decay curves could be best fitted according to the first-order relationship shown in Equation 2.


P=P<SUB>∞</SUB> <FENCE>1−e<SUP><UP>k<SUB>obs</SUB></UP> · t</SUP></FENCE> (Eq. 2)
where P and Pinfinity are the product concentrations at a given time and at infinite time, respectively, and kobs is the observed first-order rate of alkaline pH-induced enzyme inactivation (39). The influence of heparin and heparan sulfate upon the observed first-order rate cathepsins B inactivation can be described by Equation 3.
k<SUP><UP>eh</UP></SUP><SUB><UP>obs</UP></SUB>=<FR><NU>k<SUP><UP>e</UP></SUP><SUB><UP>obs</UP></SUB> (K<SUB><UP>H</UP></SUB>+&bgr; · [<UP>Hep</UP>])</NU><DE>K<SUB>H</SUB>+[<UP>Hep</UP>]</DE></FR> (Eq. 3)
where keh and ke are the observed first-order rates in presence and in absence of heparin, respectively; KH is the apparent heparin-cathepsin B dissociation constant; Hep is heparin; and beta  is the parameter of limit for kobs in presence of heparin.

Identification of Cleavage Site for Substrates-- The determination of the cleaved bonds for the peptide Abz-FRA(epsilon -Dnp)K by cathepsin B was done by HPLC and mass spectrometry analysis as described previously (33).

Circular Dichroism Spectrometry-- Circular dichroism spectra were recorded in a JASCO J-700 spectropolarimeter equipped with a stopped flow chamber and thermostated cell holder. Far ultraviolet measurements (260-200 nm) were performed at 37 °C scanning at rate of 10 nm/min on wild-type cathepsin B solution of 0.05 mg/ml in 0.05-cm cells. Circular dichroism spectra of cathepsin B-glycosaminoglycans interactions were done in 50 mM sodium phosphate buffer (pH 6.0) containing 200 mM NaCl. The observed ellipticity was normalized to units of degrees cm2/dmol. Base-line recordings in the presence or in absence of glycosaminoglycans were routinely made and used to correct cathepsin B spectra. Cathepsin B spectra were analyzed for the percentage of secondary structural elements as described previously (40).

Effect of Glycosaminoglycans on Alkaline pH-induced Unfolding of Cathepsin B-- The amount of alpha -helix structure disruption of cathepsin B induced by alkaline pH was continuously monitored by CD spectra measuring the mean residue ellipticity at 222 nm. Alkaline pH-induced denaturation of cathepsin B was performed in absence or in presence of different heparin concentrations at 37 °C in 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl. The influence of alkaline pH on alpha -helix structure of wild-type cathepsin B is a first-order relationship described by Equation 4 (39).


E<SUB><UP>helix</UP></SUB>=E<SUB>0</SUB> · e<SUP>−K<SUB><UP>unfolding</UP> · t</SUB></SUP>+R (Eq. 4)
where Ehelix is the amount of alpha -helix structures of wild-type cathepsin B at a given time of pH 8.0 exposition; E0 is cathepsin B alpha -helix content, dependent on alkaline pH, at zero time of inactivation; Kunfolding is the observed first-order rate of pH 8.0-induced unfolding of cathepsin B; and R is the residual alpha -helix content, independent of pH 8.0.

Heparin-Sepharose Affinity Chromatography-- Wild-type cathepsin B (1 µM) dissolved in 50 mM sodium phosphate buffer (pH 6.0) was applied on a heparin-Sepharose column (3 ml) previously equilibrated at 4 °C in the same buffer. A linear NaCl gradient (0-1 M) was used to elute the bonded material. The collected fractions were monitored by cathepsin B enzymatic activity upon substrate Cbz-FR-MCA.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cathepsin B Binds Heparin-Sepharose-- The interaction of cathepsin B with heparin was observed on heparin-Sepharose affinity chromatography. Cathepsin B was eluted from heparin-Sepharose column at 0.42 M of ionic strength (Fig. 1). This interaction could be disrupted specifically by the previous addition of 100 µM of heparin or heparan sulfate to cathepsin B solution. Other sulfated glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate were not capable of dislodge this binding. These results show that the binding of cathepsin B to heparin is specific and seems to be governed mainly by electrostatic interactions. These data led us to investigate the possible influence of glycosaminoglycans upon cathepsin B activity.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Cathepsin B binds heparin-Sepharose column. Cathepsin B (1 µM) dissolved in 50 mM sodium phosphate buffer (pH 6.0) was chromatographed on a heparin-Sepharose column (). Cathepsin B was preincubated with 100 µM heparin and then submitted to heparin-Sepharose column (open circle ). A linear NaCl gradient (0-1 M) was used to elute the bonded material.

The Influence of Heparin upon Cathepsin B pH Activity Profiles-- Cathepsin B is generally considered to possess both endo- and exopeptidase activity (41-43). To study the influence of heparin upon endopeptidase activity of cathepsin B, we used the substrates Cbz-FR-MCA to analyze the subsites S2 to S1 of cathepsin B. To monitor the influence of heparin upon dipeptidyl carboxypeptidase activity of cathepsin B, we used the substrate Abz-FRA(epsilon -Dnp)K, covering the cathepsin B subsites from S2 to S'2. The HPLC and mass spectrometry analysis showed that Arg-Ala is the only peptide bond cleaved by cathepsin B on substrate Abz-FRA(epsilon -Dnp)K. The presence of heparin did not change the pattern of cleavage of these peptides by cathepsin B.

The pH activity profile for the dipeptidyl carboxypeptidase activity of cathepsin B is very different from that observed for its endopeptidase activity; the maximum activity observed for the hydrolysis of substrate Abz-FRA(epsilon -Dnp)K was at pH 5.0 (Fig. 2B), whereas the pH dependence for hydrolysis of Z-FR-MCA is characterized by a gradual increase in activity when the pH is raised from 3 to 8 (Fig. 2A). The effect of heparin on pH activity profiles of wild-type human cathepsin B was analyzed by monitoring the enzyme-catalyzed hydrolysis of the fluorogenic substrates. As showed in Fig. 2, when cathepsin B was assayed with substrate Z-FR-MCA in the presence of 100 µM heparin, no significant effect on the endopeptidase pH activity profiles was observed (Fig. 2A). On the other hand, when heparin was assayed with the substrate Abz-FRA(epsilon -Dnp)K and used to monitor the dipeptidyl carboxypeptidase activity of cathepsin B (Fig. 2B), a dramatic effect of heparin was observed upon the pH activity profile. Basically, heparin promoted a general decrease in observed Abz-FRA(epsilon -Dnp)K hydrolysis second-order kcat/Km rates and shifted the cathepsin B pH activity profile about 0.5 unit to the right. This inhibition promoted by heparin is strongly dependent on pH, because no inhibition was observed when cathepsin B was assayed above pH 7. Also, the inhibition promoted by heparin at pH 5.0 is very lower than that promoted at pH 6.0. More importantly, heparin did not show effect upon E-64 cathepsin B inactivation activity (data not shown), and E-64 binds the subsites Sn of cathepsin B (44). These data suggest that the occluding loop of cathepsin B is controlling the heparin efficiency, and that the subsites S'n are involved in the interaction with heparin.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   The pH activity profiles of cathepsin B. The kinetics of Cbz-FR-MCA (A) and Abz-FRA(epsilon -Dnp)K (B) hydrolysis were performed in absence () or in the presence 100 µM heparin concentration (open circle ) at 37 °C in 50 mM sodium phosphate (pH 5.9-7.9), 50 mM citrate (pH 3.0-5.8) containing 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol.

Inhibition of the Exopeptidase Activity of Cathepsin B by Heparin-- The effect of heparin upon cathepsin B exopeptidase activity, at pH 6.0, can be described by a linear competitive type inhibition depicted in Equation 1. The influence of heparin upon substrate dissociation constant is observed in Fig. 3. Basically, heparin-cathepsin B interaction did not affect Abz-FRA(epsilon -Dnp)K catalytic constant; the presence of the substrate Abz-FRA(epsilon -Dnp)K at active site of cathepsin B excludes the binding of heparin. The results show that heparin only binds free cathepsin B with a dissociation constant of KI = 41 ± 3 µM.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of heparin on affinity of the cathepsin B for Abz-FRA(epsilon -Dnp)K. The influence of heparin concentration on cathepsin B dipeptidyl carboxypeptidase activity was determined spectrofluorometrically as described under "Experimental Procedures."

The exopeptidase activity of cathepsin B is related to the presence of two histidine residues, His110 and His111, located at occluding loop performing the S'2 subsite. His110 and His111 residues can interact with the C terminus carboxylate of a substrate (16, 34, 45). Site-directed mutagenesis has been used to investigate the importance of the His110 and His111 for the effect promoted by heparin upon dipeptidyl carboxypeptidase activity of cathepsin B. The effect of the substitution of the His110 or His111 residues by alanine can be observed in Table I. Kinetic parameters, determined using the C terminus free carboxylate substrate, Abz-FRA(epsilon -Dnp)K, showed that the substitution of His110 or His111 residues by alanine promoted a significant decrease in the peptidyldipeptidase activity of cathepsin B, as expected (45). The kcat/KM value observed for the wild-type enzyme was 1.31 × 106 M-1·s-1, and the kcat/KM value was about 6.5-fold lower for the mutant H111A and about 3.5-fold for the mutant H110A. These effects are related to the decrease in kcat; little effect in KM was observed. Surprisingly, the substitution of His111 residue by alanine was able to abolish the effect promoted by heparin upon substrate dissociation constant; the KM value was the same in the presence of 100 µM heparin (13.9 ± 0.9 µM) as in the absence of heparin (16.3 ± 0.8 µM). On the other hand, heparin showed similar effect upon mutant H110A heparin and wild-type enzyme; for both enzymes, the substrate dissociation constant was increased 3.5-fold by 100 µM heparin. These data indicate that heparin changes the dissociation constant of the cathepsin B-substrate interaction for the substrates able to reach the S'2 interaction site. The interaction of heparin with wild-type cathepsin B and H110A and H111A mutants did not affect the kcat values.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic parameters for hydrolysis of Abz-FRA (varepsilon -Dnp)K by wild-type and mutants of human cathepsin B and in the presence of heparin

Effects of Glycosaminoglycans on pH-induced Inactivation of Cathepsin B-- The deprotonation of the catalytic residue His199, catalyzed by OH- ions, is considered a crucial event for the alkaline irreversible inactivation of cathepsin B. The alkaline pH-induced inactivation, as well as the unfolding of cathepsin B, have been shown to be a first-order process (39). Fig. 4 shows the influence of heparin upon the first-order inactivation rate of cathepsin B at pH 8.0. Clearly, the data show that presence of heparin is decreasing the first-order rate of cathepsin B inactivation at pH 8.0. The kinetic parameters were determined by fitting the data to Equation 3 using nonlinear regression. The results show that heparin binds wild-type cathepsin B with a dissociation constant of KH = 18 ± 2 µM. Also, heparin induced a 5.1-fold decrease in the first-order inactivation rate of cathepsin B; the kobs value was decrease from 8.2 ± 0.6 ms-1 to 1.6 ± 0.1 ms-1 (Table II).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of heparin on pH-induced inactivation of cathepsin B. The influence of heparin concentration on kinetics of alkaline pH-induced inactivation of cathepsins B (A) were done in 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol, in the presence of 10 µM Cbz-FR-MCA at various heparin concentration: control (+) and 5 (*), 10 (black-triangle), 20 (triangle ), 40 (black-square), 80 (), 120 (), and 200 (open circle ) µM heparin. Progress of the reaction was monitored continuously by the fluorescence of the released product. B, cathepsin B first-order inactivation rate in function of heparin concentration.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Effect of heparin on the rate of inactivation of wild-type and mutants of human cathepsin B at pH 8.0 
t1/2 = ln2/kobs in the absence of heparin and teh1/2 in the presence of heparin. kobs is the observed first-order rate of alkaline pH-induced enzyme inactivation. keh and ke are observed first-order rates in presence and in absence of heparin, respectively; KH is the apparent heparin-cathepsin B dissociation constant. alpha  is the ratio of mutant and wild type cathepsin B forms dissociation constants, and beta  is the parameter of limit for kobs in presence of heparin.

The mutants H110A and H111A were also used to investigate the importance of histidine residues in the alkaline pH-induced inactivation of cathepsin B. As shown previously, the deletion of the occluding loop from cathepsin B resulted in a decrease of the pH and thermal stability of enzyme (45). Also, it has been shown that His110-Asp22 salt bridge is an important contact between the occluding loop and the central alpha -helix, and this interaction can contribute to the stability of these structural elements. On the other hand, His111 residue is not involved in electrostatic interactions with elements of the enzyme (16, 34). The effect of heparin and the substitution of the His110 or His111 residues by alanine on the alkaline pH-induced inactivation of enzymes can be observed in Table II. The data showed in Table II are consistent with these propositions. The change of His110 by alanine promoted a 4-fold decrease in the pH stability as the inactivation of the mutant H110A occurred with a t1/2 of 22 s compared with 84 s for the wild-type enzyme. On the other hand, the change of His111 by alanine had no significant effect on the enzyme stability; the inactivation of the mutant H111A occurred with a t1/2 of 75 s compared with 84 s for the wild-type enzyme. Nonetheless, the substitution of His111 residue by alanine reduced 5-fold the efficiency of heparin to protect the enzyme against alkaline pH-induced inactivation, the ratio beta /alpha observed to the wild-type enzyme was 5.1 compared with 1.0 for the mutant H111A. This effect is related to decrease the parameter beta , from 5.1 to 2.6, and increase of heparin dissociation constant (parameter alpha ), from 18 to 48 µM (Table II). However, heparin showed a similar effect upon mutant H110A and wild-type enzyme to protect these enzymes against alkaline pH-induced inactivation. In general, the effect of heparin on the first-order inactivation rate promoted by alkaline pH is comparable with the effect promoted by heparin upon dipeptidyl carboxypeptidase activity of cathepsin B (Table I), showing that the residue His111 is related to the interaction of cathepsin B with heparin.

To probe the specificity for cathepsin B interaction, other glycosaminoglycans were tested. Table III shows that, besides heparin, only heparan sulfate was able to increase the stability of cathepsin B at alkaline pH and, simultaneously, inhibited the dipeptidyl carboxypeptidase activity of cathepsin B. Other sulfated glycosaminoglycans tested, namely dermatan sulfate and chondroitin sulfate, were not able to decrease the cathepsin B affinity for the substrate or protect the enzyme stability at alkaline pH. Regarding the sulfonation of dextran sulfate, we have determined that this compound has a stronger effect than that observed for heparin on cathepsin B activity. It is possible to conclude that the electrostatic effect is important to the bind more than the structural features of the glycosaminoglycans.


                              
View this table:
[in this window]
[in a new window]
 
Table III
The second-order constant kcat/KM for the hydrolysis of Abz-FRA(varepsilon -Dnp)K and the rate of inactivation kobs at pH 8.0 of wild-type human cathepsin B in the presence of different glycosaminoglycans
kobs is the observed first-order rate of alkaline pH-induced enzyme inactivation.

In addition, we have proceeded initial experiments related to the influence of smaller fragments of heparin. The minimum size that exhibited the same effect showed by heparin was a low molecular mass heparin (4,000 Da). Heparin tetrasaccharide (Delta Idu,2Sright-arrowGlcNS,6Sright-arrowIdu, 2Sright-arrowGlcNS,6S) and disaccharide (Delta Idu,2Sright-arrowGlcNS,6S) were not able to protect the enzymatic activity against the alkaline pH inactivation. On the other hand, the oligosaccharides containing between 3 and 8 disaccharide units showed a partial effect in the protection against alkaline pH denaturation experiments (data not shown).

Effects of Heparin on Cathepsin B Circular Dichroism Spectra-- The effect of heparin on cathepsin B conformation was analyzed by CD spectroscopy. Fig. 5A show that the presence of 100 µM heparin causes a significant change in the spectral envelope of the cathepsin B, leading a decrease of the ellipticity value at [theta ]222 nm. These data show that heparin increases the helicity of cathepsin B, suggesting that this change is due to cathepsin B heparin interaction. As expected, the interaction between cathepsin B and heparin was disrupted by the addition of 0.5 M NaCl (Fig. 5B). The spectrum obtained in the presence of 0.5 M NaCl is very similar to the spectrum obtained for the cathepsin B alone in the presence of 0.5 M NaCl.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of heparin on cathepsin B circular dichroism spectra. A, cathepsin B CD spectra were determined at pH 6.0 as described under "Experimental Procedures." The CD spectra were performed in absence of heparin () or in the presence of 100 µM heparin (black-triangle). B, the cathepsin B circular dichroism spectra were obtained at 0.5 M NaCl in the absence () or in the presence (open circle ) of 100 µM heparin.

The Influence of Heparin upon Unfolding of Cathepsin B Induced by Alkaline pH-- Inactivation of human cathepsin B is accompanied by large structural changes and highly dependent on pH, temperature, and ionic strength of the medium (39). The break of the thiolate-imidazolium ion pair, catalyzed by alkaline pH, as well as the disruption of other important salt bridges, such as Asp22-His110 and Arg116-Asp224, located at the occluding loop (16, 34), and two interdomain bonds, Asp40-Arg202 and Arg41-Glu163, (16, 39) are responsible for the destabilization of the cathepsin B central alpha -helix, which contains the active site Cys29 residue. Fig. 6A shows the measurements of circular dichroism of cathepsin B at pH 5.5 and 8.0. The results show that a drastic increase in the ellipticity values was observed when cathepsin B was exposed at pH 8.0, indicating a large loss of the native structure. Table IV summarizes the secondary structure content of cathepsin B in presence or in absence of heparin at different pH levels. A large decrease in the alpha -helix content was observed at pH 8.0, whereas the beta -sheet contents increases. At pH 5.5, the alpha -helix and beta -sheet contents were 34.2 and 21%, respectively, and at pH 8.0, the alpha -helix and beta -sheet contents were 14.6 and 35.5%, respectively. However, when cathepsin B was preincubated with 100 µM heparin, the amount of structure unfolding, induced by alkaline pH, was largely decreased.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Heparin prevents the loss of cathepsin B alpha -helix structures induced by alkaline pH. A, the CD analysis of cathepsin B were proceeded at pH 5.5 () and 8.0 (black-square), and pH 8.0 in the presence () of 100 µM heparin. Other experimental details were as described under "Experimental Procedures." B, the rate of loss of cathepsin B alpha -helix structures at pH 8.0 in absence () or in presence (open circle ) of 100 µM heparin.


                              
View this table:
[in this window]
[in a new window]
 
Table IV
The influence of heparin upon far UV (200-260 nm) CD spectra of wild-type human cathepsin B at pH 5.5 and pH 8.0

In an attempt to compare rates of the inactivation and of unfolding of cathepsin B, the conformational change of cathepsin B, induced by alkaline pH, were monitored continuously by circular dichroism at 222 nm (Fig. 6B). The alkaline pH-induced unfolding of cathepsin B is shown to be a first-order process (39). The experimental curves were fitted to Equation 4, and the first-order constants were determined; in absence of heparin Kunfolding = 7.8 ± 0.4 ms-1, and in the presence of 100 µM heparin the observed constant was decreased 3.1-fold (Kunfolding = 2.5 ± 0.2 ms-1). The rate constants of conformational change of cathepsin B were very similar to results show in Fig. 4; in absence of heparin kobs = 8.2 ± 0.6 ms-1, and in presence 100 µM heparin kobs = 2.6 ± 0.2 ms-1, suggesting that both heparin effects are linked.

Activity of Cathepsin B toward Azocasein Substrate-- The experiments with azocasein showed that heparin does not exert influence in the endopeptidase activity of cathepsin B cleaving protein substrates. In addition, it was also possible to observe that heparin protects cathepsin B from alkaline pH denaturation in the cleavage of azocasein, used as a model for protein substrates (Fig. 7).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Protection against pH alkaline inactivation of cathepsin B promoted by heparin in the cleavage of azocasein. Preactivated cathepsin B was preincubated at pH 8.0 for the times indicated at 37 °C and then assayed for endopeptidase activity using azocasein, in the absence (open circle ) or in the presence () of heparin (20 µM).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We observed that cathepsin B interacted with heparin-Sepharose resin (Fig. 1). Cathepsin B is generally considered to possess both endo- and exopeptidase activity (41-43, 45). The binding of heparin to cathepsin B did not perturb its endopeptidase activity upon the fluorogenic substrate Z-FR-MCA (Fig. 2A) or upon azocasein used as a model for a protein substrate (Fig. 7). Also, heparin binding was not capable of counteracting the inhibitory activity of E-64 (data not shown). Because Z-FR-MCA and E-64 interact with cathepsin B at S1 and S2 subsites (16, 34, 45), we can conclude that heparin binding to cathepsin B does not affect Sn subsites of the enzyme. On the other hand, it was observed that heparin inhibits cathepsin B exopeptidase activity (Fig. 2B). The inhibition promoted by heparin upon dipeptidyl carboxypeptidase activity of cathepsin B strongly suggests that heparin interacts with cathepsin B at the occluding loop region and contains residues His110 and His111 at subsites S'2 and S'3 that can interact with the lysin C-terminal carboxylate of the substrate Abz-FRA(epsilon -Dnp)K (16, 41, 45).

The same above effect was then assessed by site-directed mutagenesis studies. Indeed, as shown in Table I, the mutant H111A was not inhibited by heparin, but the mutant H110A was inhibited by heparin at the same extension as the wild-type enzyme was. These data clearly show the main role of His111 on the interaction between heparin and cathepsin B.

It has been shown that the deletion of the occluding loop from cathepsin B results in a decrease of the pH and thermal stability of enzyme (45). It was also observed that His110-Asp22 salt bridge is an important contact between the occluding loop and the central alpha -helix, and this interaction can contribute to the stability of these structural elements (16, 34, 46). On the other hand, His111 residue is not involved in electrostatic interactions with elements of the enzyme (16, 34). The mutants H110A and H111A were also used to investigate the importance of histidine residues in the alkaline pH-induced inactivation of cathepsin B. Table II shows that the efficiency of heparin to protect cathepsin B against alkaline pH-induced inactivation is very dependent on His111. These results show that the effect of heparin on the first-order inactivation rate promoted by alkaline pH is comparable with the effect promoted by heparin upon dipeptidyl carboxypeptidase activity of cathepsin B (Table I). The poor inhibition of membrane-bound forms of cathepsin B by compounds like CA-030, which requires interactions with His110 and His111 residues, is probably related to the interactions of the enzyme with heparan sulfate from the cell surface (34).

The interaction between cathepsin B and heparin or heparan sulfate is specific, as other sulfated glycosaminoglycans, namely chondroitin sulfate and dermatan sulfate, were not able to inhibit the dipeptidyl carboxypeptidase activity of cathepsin B or induce protection against alkaline pH inactivation (Table III). On the other hand, dextran sulfate, a more sulfonated polymer, has a stronger effect than heparin on cathepsin B activity. It is possible to conclude that the electrostatic effect is more important to the binding than the structural features of glycosaminoglycans.

The interaction of heparin with cathepsin B can be monitored by CD spectroscopy analysis. Fig. 5A shows that heparin significantly decreases the molar ellipticity of the cathepsin B CD spectra at [theta ]222 nm, showing that the presence of heparin increases the alpha -helix content of the enzyme. As expected, this effect was dependent on ionic strength. Addition of 0.5 M NaCl to heparin-cathepsin B solution causes a spectral change consistent with the disruption of the heparin-cathepsin B complex (Fig. 5B).

As already shown in the literature (39), a dramatic increase in the ellipticity molar value at [theta ]222 nm was detected at pH 8.0, suggesting a large decrease in the alpha -helix content of the enzyme at alkaline pH (Fig. 6A). However, when cathepsin B was preincubated with heparin, the amount of alpha -helix structure disruption induced by alkaline pH was decreased (Table IV). The Fig. 6B shows that the rate of unfolding of cathepsin B at pH 8.0 was decreased by the presence of heparin at the same extension that first-order inactivation rate of cathepsin B at alkaline pH was.

The effect of heparin on the rate of unfolding of cathepsin B at pH 8.0 (Fig. 6B) is comparable with the first-order inactivation rate promoted by alkaline pH (Table II) that by its turn is related to the inhibition of heparin upon the dipeptidyl carboxypeptidase activity of cathepsin B (Table I). Most of these effects are related to the interaction of heparin with His111 residue of the cathepsin B occluding loop. Taken together, these results show that, in all cases, heparin binding is perturbing cathepsin B in a similar manner.

The presence of cathepsin B at the plasma membrane results in focal dissolution of extracellular matrix proteins and enables the tumor cell to invade the tissue (14, 17-20). Our results suggest that the cell surface heparan sulfate can anchor the membrane forms of cathepsin B, and such complexation affects the cathepsin B activities. The coupling of cathepsin B with heparan sulfate increases its half-life 5-fold (t1/2) at physiological pH and, quite probably, potentiates the endopeptidase activity of the enzyme at the cell surface. In addition, it was also possible to observe that cathepsin B is protected by heparin from alkaline pH denaturation in the cleavage of protein substrates (Fig. 7). The endopeptidase activity of cathepsin B is related to the degradation of extracellular matrix proteins (19, 20). These results are in agreement with the observation that the membrane-bound forms of cathepsin B are very resistant to inactivation at neutral pH (32). As previously mentioned, the mechanism of secretion and insertion of cathepsin B on the plasma membrane are not fully understood (27, 29). Mannose-6-phosphate-independent targeting has been proposed for cathepsin B. So, according to this scenario, the cell surface heparan sulfate proteoglycans can be anchoring a pool of the membrane forms of cathepsin B.

Recently it has been shown that cathepsin B colocalizes with annexin II tetramer on the surface of tumor cells (47). In addition, annexin II tetramer was also shown to bind heparin with high affinity dissociation constant (Kd = 32 nM) (48). These results and our present data strongly suggest that heparan sulfate and annexin II tetramer together can act as an important binding site for cathepsin B on the cell surface. Moreover, the cell surface heparan sulfate proteoglycans are in a constant turnover, as a result of its continuous secretion and endocytosis (49-51). It has been shown that some proteins that are bound to heparan sulfate glycosaminoglycans chains are endocytosed together with proteoglycans, e.g. fibroblast growth factor (52), trombospodin (53), and lipoprotein lipases (54). It is interesting to observe that in the lysosomal vesicles there is a high concentration of cathepsin B (26) and that the heparan sulfate is also present in this compartment during its intracellular traffic (49-51). These observations suggest that the mechanism of insertion of cathepsin B on the plasma membrane and its cellular traffic can be dependent on heparan sulfate proteoglycans present at cell surface. In addition, this hypothesis is also supported by the perinuclear cathepsin B location in tumor cells (23, 26, 55), as also observed for the cellular distribution of heparan sulfate complexed to fibroblast growth factor (52). This intracellular location of cathepsin B may play a role in nuclear functions, becoming a part of the dramatic phenotypic transformation, known as "activation," observed in carcinogenic process (56).

Acknowledgments-- We thank Drs. Robert Ménard (Biotechnology Research Institute, Montréal, Québec, Canada) and John S. Mort (McGill University, Montréal, Québec, Canada) for supplying the wild type and mutants of cathepsin B and Dr. Michel Goldberg (Institut Pasteur, Paris, France) for helping in 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, SP, 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.

Dagger Dagger To whom correspondence should be addressed. Tel.: 55-11-4798-7102; E-mail: ivarne@ccb.umc.br.

Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M003820200


    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-methyl-coumarinyl-7-amide; Abz-FRA(epsilon -Dnp)K, ortho-aminobenzoyl-L-phenylalanyl-L-argininyl-L-alanyl-L-lysyn epsilon -N-2,4-dinitrophenylamide; HPLC, high pressure liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Dietrich, C. P., Nader, H. B., and Straus, H. A. (1983) Biochem. Biophys. Res. Commun. 111, 865-871[Medline] [Order article via Infotrieve]
2. Dietrich, C. P., Tersariol, I. L. S., Toma, L., Moraes, C. T., Porcionatto, M. A., Oliveira, F. W., and Nader, H. B. (1998) Cell. Mol. Biol. 44, 417-429
3. Yanagishita, M., and Hascall, V. C. (1992) J. Biol. Chem. 267, 9451-9454[Free Full Text]
4. Elenius, K., and Jalkanen, M. (1994) J. Cell Sci. 107, 2975-2982[Free Full Text]
5. David, G. (1993) FASEB J. 7, 1023-1030[Abstract/Free Full Text]
6. Iozzo, R., Cohen, I. R., Grässel, S., and Murdock, A. D. (1995) Biochem. J. 302, 625-629
7. Conrad, H. E. (1998) Heparin-binding Proteins , Academic Press Inc., New York
8. Gettins, P. G. W., Patston, P. A., and Olson, S. T. (1996) Serpins: Structure, Function, and Biology , R. G. Landes Co., Austin, TX
9. Ermolieff, J., Boudier, C., Laine, A., Meyer, B., and Bieth, J. G. (1994) J. Biol. Chem. 269, 29502-29508[Abstract/Free Full Text]
10. Fath, M. A., Wu, X., Hileman, R. E., Linhardt, R. J., Kashem, M. A., Nelson, R. M., Wright, C. D., and Abraham, W. (1998) J. Biol. Chem. 273, 13563-3569[Abstract/Free Full Text]
11. Kainulainen, V., Wang, H., Schick, C., and Bernfield, M. (1998) J. Biol. Chem. 273, 11563-11569[Abstract/Free Full Text]
12. Katunuma, N. (1989) Intracellular Proteolysis , Japan Scientific Societies Press, Tokyo, Japan
13. Del Nery, E., Juliano, M. A., Lima, A. P. C. A., Scharfstein, J., and Juliano, L. (1997) J. Biol. Chem. 272, 25713-25718[Abstract/Free Full Text]
14. Sloane, B. F., Rozhin, J., Johnson, K., Taylor, H., Crissman, J. D., and Honn, K. V. (1986) Proc. Natl. Acad. Sci. 83, 2483-2487[Abstract]
15. Turk, B., Turk, V., and Turk, D. (1997) Biol. Chem. Hoppe-Seyler 378, 141-150
16. Musil, D., Zucic, D., Turk, D., Engh, R. A., Mayr, I., Huber, R., Popovic, T., Turk, V., Towatari, T., Katuma, N., and Bode, W. (1991) EMBO J. 10, 2321-2330[Abstract]
17. Buck, M. R., Karustis, D. G., Day, N. A., Honn, K. V., and Sloane, B. F. (1992) Biochem. J. 282, 273-278[Medline] [Order article via Infotrieve]
18. Sloane, B. F. (1996) Nat. Biotechnol. 14, 826-827[Medline] [Order article via Infotrieve]
19. Weiss, R. E., Liu, B. C. S., Ahlering, T., Dubeau, l., and Droller, M. J. (1990) Br. J. Urol. 144, 798-804
20. Koblinski, J. E., and Sloane, B. F. (1997) Medical Aspects of Protease and Protease Inhibitors , pp. 185-193, IOS Press, Amsterdam, The Netherlands
21. Brown, W. J., Goodhouse, J., and Farquhar, M. G. (1986) J. Cell Biol. 110, 1235-1247
22. Kasper, D., Dittmer, F., von Figura, K., and Pohlmann, R. (1996) J. Cell Biol. 134, 615-623[Abstract]
23. Iacobuzio-Donahue, C. A., Shuja, S., Cai, J., Peng, P., and Murnane, M. J. (1997) J. Biol. Chem. 272, 29190-29199[Abstract/Free Full Text]
24. Hanewinkel, H., Gloss, J., and Kresse, H. (1987) J. Biol. Chem. 262, 12351-12355[Abstract/Free Full Text]
25. De Stefanis, D., Demoz, M., Dragonetti, A., Houri, J. J., Ogier-Denis, E., Codogno, P., Baccino, F. M., and Isidoro, C. (1997) Cell Tissue Res. 289, 109-117[CrossRef][Medline] [Order article via Infotrieve]
26. Kos, J., and Lah, T. T. (1998) Oncol. Rep. 5, 1349-1361[Medline] [Order article via Infotrieve]
27. Kornfeld, S. (1990) Biochem. Soc. Trans. 18, 367-374[Medline] [Order article via Infotrieve]
28. Mort, J. S., and Recklies, A. D. (1986) Biochem. J. 233, 57-63[Medline] [Order article via Infotrieve]
29. Linebaugh, B. E., Sameni, M., Day, N. A., Sloane, B. F., and Keppler, D. (1999) Eur. J. Biochem. 264, 100-109[Abstract/Free Full Text]
30. Mach, L., Mort, J. S., and Glössl, J. (1994) J. Biol. Chem. 269, 13030-13035[Abstract/Free Full Text]
31. Jerala, R., Zerovnic, E., Kidric, J., and Turk, V. (1998) J. Biol. Chem. 273, 11498-11504[Abstract/Free Full Text]
32. Sloane, B. F., Rozhin, J., Lah, T. T., Day, N. A., Buck, M., Ryan, R. E., Crissman, J. D., and Honn, K. V. (1988) Adv. Exp. Med. Biol. 233, 259-268[Medline] [Order article via Infotrieve]
33. Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Júdice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438[Abstract/Free Full Text]
34. Nägler, D. K., Storer, A. C., Portaro, F. C. V., Carmona, E., Juliano, L., and Ménard, R. (1997) Biochemistry 36, 12608-12615[CrossRef][Medline] [Order article via Infotrieve]
35. Ménard, R., Khouri, H. E., Plouffe, C., Dupras, R., Ripoll, D., Vernet, T., Tessier, D. C., Laliberté, F., Thomas, D. Y., and Storer, A. C. (1990) Biochemistry 29, 6706-6713[Medline] [Order article via Infotrieve]
36. Hirata, I. Y., Cezari, M. H. S., Nakaie, C. R., Boschcov, P., Ito, A. S., Juliano, M., and Juliano, L. (1994) Lett. Peptide Sci. 1, 299-308
37. Chagas, J. R., Juliano, L., and Prado, E. S. (1991) Anal. Biochem. 192, 419-425[Medline] [Order article via Infotrieve]
38. Starkey, P. M. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed) , pp. 57-89, Elvesier/North Holland, Amsterdam, The Netherlands
39. Turk, B., Dolenc, I., &Zbreve;erovnic, E., Turk, D., Gubensek, F., Turk, V. (1994) Biochemistry 33, 14800-14806[Medline] [Order article via Infotrieve]
40. Sreerama, N., and Woody, R. W. (1993) Anal. Biochem. 209, 32-44[CrossRef][Medline] [Order article via Infotrieve]
41. Barrett, A. J., and Kirschke, H. (1981) Methods Enzymol. 80, 535-561[Medline] [Order article via Infotrieve]
42. Koga, H., Yamada, H., Nishimura, Y., and Imoto, T. (1991) J. Biochem. (Tokyo) 110, 179-188[Abstract]
43. Nägler, D. R., Tam, W., Storer, A. C., Krupa, J. C., Mort, J. S., and Ménard, R. (1999) Biochemistry 38, 4868-4874[CrossRef][Medline] [Order article via Infotrieve]
44. Katunuma, N., and Kominami, E. (1995) Methods Enzymol. 251, 382-397[Medline] [Order article via Infotrieve]
45. Illy, C., Quraishi, O., Wang, J., Purisima, E., Vernet, T., and Mort, J. S. (1997) J. Biol. Chem. 272, 1197-1202[Abstract/Free Full Text]
46. Quraishi, O., Nägler, D. R., Fox, T., Sivaraman, J., Cygler, M., Mort, J. S., and Store, A. C. (1999) Biochemistry 38, 5017-5023[CrossRef][Medline] [Order article via Infotrieve]
47. Mai, J., Finley Jr, R. L., Waisman, D. M., and Sloane, B. F. (2000) J. Biol. Chem. 275, 12806-12812[Abstract/Free Full Text]
48. Kassam, G., Manro, A., Braat, C. E., Louie, P., Fitzpatrick, S. L., and Waisman, D. M. (1997) J. Biol. Chem. 272, 15093-15100[Abstract/Free Full Text]
49. Kramer, P. M. (1971) Biochemistry 10, 1437-1445[Medline] [Order article via Infotrieve]
50. Bienkowiski, M. J., and Conrad, H. E. (1984) J. Biol. Chem. 259, 12989-12996[Abstract/Free Full Text]
51. Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10260-10269[Abstract/Free Full Text]
52. Reiland, J., and Rapraeger, A. C. (1993) J. Cell Sci. 105, 1085-1093[Abstract/Free Full Text]
53. Mikhailenko, I., Kounnas, M. Z., and Strickland, D. K. (1995) J. Biol. Chem. 270, 9543-9549[Abstract/Free Full Text]
54. Jackson, R. I., Busch, S. J., and Cardin, A. D. (1991) Physiol. Rev. 71, 481-539[Free Full Text]
55. Yan, S., Sameni, M., and Sloane, B. F. (1998) Biol. Chem. Hoppe-Seyler 379, 113-123
56. Bahr, M. J., Vincent, K. J., Arthur, M. J., Fowler, A. V., Smart, D. E., Wright, M. C., Clark, I. M., Benyon, R. C., Iredale, J. P., and Mann, D. A. (1999) Hepatology 29, 839-848[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.