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Cathepsin B Activity Regulation

HEPARIN-LIKE GLYCOSAMINOGLYCANS PROTECT HUMAN CATHEPSIN B FROM ALKALINE pH-INDUCED INACTIVATION*
Open AccessPublished:January 12, 2001DOI:https://doi.org/10.1074/jbc.M003820200
      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 α-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 α-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 (t12) 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.
      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(ε-Dnp)K
      ortho-aminobenzoyl-l-phenylalanyl-l-argininyl-l-alanyl-l-lysyn ε-N-2,4-dinitrophenylamide
      HPLC
      high pressure liquid chromatography
      Heparan sulfate is an ubiquitous glycosaminoglycan of animal cells (
      • Dietrich C.P.
      • Nader H.B.
      • Straus H.A.
      ). These classes of compounds are heteropolysaccharides made up of repeating units of disaccharides, an uronic acid residue, eitherd-glucuronic acid or l-iduronic acid, andd-glucosamine with N- and 6-O-sulfates and N-acetyl substitutions (
      • Dietrich C.P.
      • Tersariol I.L.S.
      • Toma L.
      • Moraes C.T.
      • Porcionatto M.A.
      • Oliveira F.W.
      • Nader H.B.
      ). 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 (
      • Yanagishita M.
      • Hascall V.C.
      ,
      • Elenius K.
      • Jalkanen M.
      ), and the glypican family is anchored by glycosilyl phosphatidylinositol-anchor core proteins (
      • David G.
      ). Also, heparan sulfate proteoglycans are present in basement membranes performing the perlecan family (
      • Iozzo R.
      • Cohen I.R.
      • Grässel S.
      • Murdock A.D.
      ).
      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 (
      • Conrad H.E.
      ). 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 (
      • Gettins P.G.W.
      • Patston P.A.
      • Olson S.T.
      ,
      • Ermolieff J.
      • Boudier C.
      • Laine A.
      • Meyer B.
      • Bieth J.G.
      ,
      • Fath M.A.
      • Wu X.
      • Hileman R.E.
      • Linhardt R.J.
      • Kashem M.A.
      • Nelson R.M.
      • Wright C.D.
      • Abraham W.
      ,
      • Kainulainen V.
      • Wang H.
      • Schick C.
      • Bernfield M.
      ). 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 (
      • Katunuma N.
      ), parasite infection (
      • Del Nery E.
      • Juliano M.A.
      • Lima A.P.C.A.
      • Scharfstein J.
      • Juliano L.
      ), and tumor metastasis (
      • Sloane B.F.
      • Rozhin J.
      • Johnson K.
      • Taylor H.
      • Crissman J.D.
      • Honn K.V.
      ). Cathepsin B shows close structural homology to the other cysteine proteases of the papain family (
      • Turk B.
      • Turk V.
      • Turk D.
      ). 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 (
      • Musil D.
      • Zučič D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovič T.
      • Turk V.
      • Towatari T.
      • Katuma N.
      • Bode W.
      ).
      It has been shown that lysosomal cysteine proteinases, specially cathepsin B, can participate in tumor invasion by degradation of extracellular matrix components (
      • Buck M.R.
      • Karustis D.G.
      • Day N.A.
      • Honn K.V.
      • Sloane B.F.
      ). This can take place either intracellularly by heterophagosomal activity of tumors cell (
      • Sloane B.F.
      ) or extracellularly by cell surface associated cathepsin B (
      • Sloane B.F.
      • Rozhin J.
      • Johnson K.
      • Taylor H.
      • Crissman J.D.
      • Honn K.V.
      ). 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 (
      • Weiss R.E.
      • Liu B.C.S.
      • Ahlering T.
      • Dubeau L.
      • Droller M.J.
      ,
      • Koblinski J.E.
      • Sloane B.F.
      ). Trafficking and targeting of lysosomal enzymes is mostly mediated by mannose-6-phosphate receptor pathways (
      • Brown W.J.
      • Goodhouse J.
      • Farquhar M.G.
      ). 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 (
      • Kasper D.
      • Dittmer F.
      • von Figura K.
      • Pohlmann R.
      ) or by changes in glycosylation pattern of lysosomal enzymes as observed for cathepsin B in carcinoma cells (
      • Iacobuzio-Donahue C.A.
      • Shuja S.
      • Cai J.
      • Peng P.
      • Murnane M.J.
      ). Indeed, mannose-6-phosphate-independent targeting has been proposed for cathepsin B in normal cell (
      • Hanewinkel H.
      • Gloss J.
      • Kresse H.
      ) and human colon carcinoma cell lines (
      • De Stefanis D.
      • Demoz M.
      • Dragonetti A.
      • Houri J.J.
      • Ogier-Denis E.
      • Codogno P.
      • Baccino F.M.
      • Isidoro C.
      ). 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 (
      • Iacobuzio-Donahue C.A.
      • Shuja S.
      • Cai J.
      • Peng P.
      • Murnane M.J.
      ). 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 (
      • Kos J.
      • Lah T.T.
      ).
      The mechanism of secretion and insertion of cathepsin B at the plasma membrane are not fully understood (
      • Kornfeld S.
      ). Cathepsin B is secreted by normal and by tumor cells as the precursor forms (
      • Mort J.S.
      • Recklies A.D.
      ), whereas many types of tumors cells may also release mature, active form of cathepsin B (
      • Linebaugh B.E.
      • Sameni M.
      • Day N.A.
      • Sloane B.F.
      • Keppler D.
      ). 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 (
      • Mach L.
      • Mort J.S.
      • Glössl J.
      ,
      • Jerala R.
      • Zerovnic E.
      • Kidric J.
      • Turk V.
      ). 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 (
      • Turk B.
      • Turk V.
      • Turk D.
      ). On the other hand, it has been shown that membrane-bound forms of cathepsin B are very resistant to inactivation at neutral pH (
      • Sloane B.F.
      • Rozhin J.
      • Lah T.T.
      • Day N.A.
      • Buck M.
      • Ryan R.E.
      • Crissman J.D.
      • Honn K.V.
      ).
      Recently, we have shown that heparin and heparan sulfate bind papain specifically; this interaction induces an increase of α-helix content of papain, which 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.
      • Tersariol I.L.S.
      ). 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.

      DISCUSSION

      We observed that cathepsin B interacted with heparin-Sepharose resin (Fig. 1). Cathepsin B is generally considered to possess both endo- and exopeptidase activity (
      • Barrett A.J.
      • Kirschke H.
      ,
      • Koga H.
      • Yamada H.
      • Nishimura Y.
      • Imoto T.
      ,
      • Nägler D.R.
      • Tam W.
      • Storer A.C.
      • Krupa J.C.
      • Mort J.S.
      • Ménard R.
      ,
      • Illy C.
      • Quraishi O.
      • Wang J.
      • Purisima E.
      • Vernet T.
      • Mort J.S.
      ). The binding of heparin to cathepsin B did not perturb its endopeptidase activity upon the fluorogenic substrate Z-FR-MCA (Fig. 2 A) 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 (
      • Musil D.
      • Zučič D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovič T.
      • Turk V.
      • Towatari T.
      • Katuma N.
      • Bode W.
      ,
      • Nägler D.K.
      • Storer A.C.
      • Portaro F.C.V.
      • Carmona E.
      • Juliano L.
      • Ménard R.
      ,
      • Illy C.
      • Quraishi O.
      • Wang J.
      • Purisima E.
      • Vernet T.
      • Mort J.S.
      ), 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. 2 B). 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 S2 and S3 that can interact with the lysin C-terminal carboxylate of the substrate Abz-FRA(ε-Dnp)K (
      • Musil D.
      • Zučič D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovič T.
      • Turk V.
      • Towatari T.
      • Katuma N.
      • Bode W.
      ,
      • Barrett A.J.
      • Kirschke H.
      ,
      • Illy C.
      • Quraishi O.
      • Wang J.
      • Purisima E.
      • Vernet T.
      • Mort J.S.
      ).
      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 (
      • Illy C.
      • Quraishi O.
      • Wang J.
      • Purisima E.
      • Vernet T.
      • Mort J.S.
      ). It was also observed that His110-Asp22 salt bridge is an important contact between the occluding loop and the central α-helix, and this interaction can contribute to the stability of these structural elements (
      • Musil D.
      • Zučič D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovič T.
      • Turk V.
      • Towatari T.
      • Katuma N.
      • Bode W.
      ,
      • Nägler D.K.
      • Storer A.C.
      • Portaro F.C.V.
      • Carmona E.
      • Juliano L.
      • Ménard R.
      ,
      • Quraishi O.
      • Nägler D.R.
      • Fox T.
      • Sivaraman J.
      • Cygler M.
      • Mort J.S.
      • Store A.C.
      ). On the other hand, His111 residue is not involved in electrostatic interactions with elements of the enzyme (
      • Musil D.
      • Zučič D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovič T.
      • Turk V.
      • Towatari T.
      • Katuma N.
      • Bode W.
      ,
      • Nägler D.K.
      • Storer A.C.
      • Portaro F.C.V.
      • Carmona E.
      • Juliano L.
      • Ménard R.
      ). 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 (
      • Nägler D.K.
      • Storer A.C.
      • Portaro F.C.V.
      • Carmona E.
      • Juliano L.
      • Ménard R.
      ).
      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. 5 A shows that heparin significantly decreases the molar ellipticity of the cathepsin B CD spectra at [θ]222 nm, showing that the presence of heparin increases the α-helix content of the enzyme. As expected, this effect was dependent on ionic strength. Addition of 0.5m NaCl to heparin-cathepsin B solution causes a spectral change consistent with the disruption of the heparin-cathepsin B complex (Fig. 5 B).
      As already shown in the literature (
      • Turk B.
      • Dolenc I.
      • Z̆erovnic E.
      • Turk D.
      • Gubenšek F.
      • Turk V.
      ), a dramatic increase in the ellipticity molar value at [θ]222 nm was detected at pH 8.0, suggesting a large decrease in the α-helix content of the enzyme at alkaline pH (Fig. 6 A). However, when cathepsin B was preincubated with heparin, the amount of α-helix structure disruption induced by alkaline pH was decreased (Table IV). The Fig. 6 Bshows 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. 6 B) 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 (
      • Sloane B.F.
      • Rozhin J.
      • Johnson K.
      • Taylor H.
      • Crissman J.D.
      • Honn K.V.
      ,
      • Buck M.R.
      • Karustis D.G.
      • Day N.A.
      • Honn K.V.
      • Sloane B.F.
      ,
      • Sloane B.F.
      ,
      • Weiss R.E.
      • Liu B.C.S.
      • Ahlering T.
      • Dubeau L.
      • Droller M.J.
      ,
      • Koblinski J.E.
      • Sloane B.F.
      ). 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 (t12) 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 (
      • Weiss R.E.
      • Liu B.C.S.
      • Ahlering T.
      • Dubeau L.
      • Droller M.J.
      ,
      • Koblinski J.E.
      • Sloane B.F.
      ). These results are in agreement with the observation that the membrane-bound forms of cathepsin B are very resistant to inactivation at neutral pH (
      • Sloane B.F.
      • Rozhin J.
      • Lah T.T.
      • Day N.A.
      • Buck M.
      • Ryan R.E.
      • Crissman J.D.
      • Honn K.V.
      ). As previously mentioned, the mechanism of secretion and insertion of cathepsin B on the plasma membrane are not fully understood (
      • Kornfeld S.
      ,
      • Linebaugh B.E.
      • Sameni M.
      • Day N.A.
      • Sloane B.F.
      • Keppler D.
      ). 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 (
      • Mai J.
      • Finley Jr R.L.
      • Waisman D.M.
      • Sloane B.F.
      ). In addition, annexin II tetramer was also shown to bind heparin with high affinity dissociation constant (Kd = 32 nm) (
      • Kassam G.
      • Manro A.
      • Braat C.E.
      • Louie P.
      • Fitzpatrick S.L.
      • Waisman D.M.
      ). 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 (
      • Kramer P.M.
      ,
      • Bienkowiski M.J.
      • Conrad H.E.
      ,
      • Yanagishita M.
      • Hascall V.C.
      ). 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 (
      • Reiland J.
      • Rapraeger A.C.
      ), trombospodin (
      • Mikhailenko I.
      • Kounnas M.Z.
      • Strickland D.K.
      ), and lipoprotein lipases (
      • Jackson R.I.
      • Busch S.J.
      • Cardin A.D.
      ). It is interesting to observe that in the lysosomal vesicles there is a high concentration of cathepsin B (
      • Kos J.
      • Lah T.T.
      ) and that the heparan sulfate is also present in this compartment during its intracellular traffic (
      • Kramer P.M.
      ,
      • Bienkowiski M.J.
      • Conrad H.E.
      ,
      • Yanagishita M.
      • Hascall V.C.
      ). 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 (
      • Iacobuzio-Donahue C.A.
      • Shuja S.
      • Cai J.
      • Peng P.
      • Murnane M.J.
      ,
      • Kos J.
      • Lah T.T.
      ,
      • Yan S.
      • Sameni M.
      • Sloane B.F.
      ), as also observed for the cellular distribution of heparan sulfate complexed to fibroblast growth factor (
      • Reiland J.
      • Rapraeger A.C.
      ). 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 (
      • 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.
      • Mann D.A.
      ).

      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.

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