Inactivation of blood plasma alpha 1-proteinase inhibitor as affected by 2 splenic thiol proteinases active in a neutral medium

It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.     

Acid-stable low molecular mass proteinase inhibitors in human lung lavage

An acid-stable, low molecular mass proteinase inhibitor, bronchial mucus proteinase inhibitor (BMPI), has been isolated from sputum and partially characterised. A single band with a modal molecular mass of 18 700 was observed following SDS-polyacrylamide gel electrophoresis. BMPI inhibited human leukocyte elastase, cathepsin G, trypsin and chymotrypsin, but not porcine pancreatic elastase. Although BMPI had a molecular mass close to the similarly isolated inhibitor of Girard et al. (Girard, F., Tournier, J.M., Polu, J.M. & Sadoul, P. (1980), Bull. Eur. Physiopathol. Respir. 16 (Suppl.) 237-245), and although it showed immunological cross reactivity to the low molecular mass inhibitor of Kramps et al. (Kramps, J.A., Franken, C., Meyer, C.J.L.M. & Dijkman, J.H. (1981) J. Histochem. Cytochem. 29, 712-719), it was found to have an amino-acid profile different to any previously described inhibitor. BMPI was detectable in bronchoalveolar lavage fluid collected from healthy and diseased human lungs. The median molar ratio of BMPI/alpha 1-proteinase inhibitor (alpha 1 PI) observed in these lavage samples was 0.7, which is generally higher than those derived from the data of other authors. This suggests that BMPI is a different protein to those previously described, although its exact relationship to other low molecular mass proteinase inhibitors remains to be determined.     

Purification and characterization of cathepsin J from rat liver.

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecular-mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (alpha subunit) was a glycoprotein with a molecular mass of 19-24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (beta subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine beta-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3- methylbutylamide (E-64-c) was 1800 nM, which was 100-500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl-aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.     

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.     

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.     

Studies on cathepsin B in human articular cartilage.

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.     

组织蛋白酶B的新型天然抑制剂

组织蛋白酶B是木瓜蛋白酶类半胱氨酸蛋白酶家族的重要成员,它与人类多种疾病相关,尤其是在恶性肿瘤的侵袭转移过程中扮演了重要角色.通过随机筛选,发现了五个对组织蛋白酶B具有较好抑制活性的天然化合物prodelphinidin B-23'-O-gallate(1),prodelphinidin B-2(2),ImJcyarddin B-2(3),puexin A(4)和(-)epigallocatechin-3-O-gallate(5),其IC50值分别为0.58,0.44,0.76,2.07和0.96umol/L.这五个抑制剂为黄烷醇类化合物,均为组织蛋白酶B的新型天然抑制剂.     

Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds

Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate. HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 nmol/1 to 8.0 nmol/1. Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of alpha-naphthyl acetate esterase and reduced cell division. Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells. Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 micrograms/ml endotoxin (lipopolysaccharide) from Escherichia coli, but not carrageenan. The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 mumol/1 dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin. The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes.     

Proteinase expression during differentiation of human osteoclasts in vitro

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.     

Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.     

Nucleo-cytoplasmic relationships of oestrogen receptors in rat liver during the oestrous cycle and in response to administered natural and synthetic oestrogen.

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

Human cathepsin G. Catalytic and immunological properties.

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.     

Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts

Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly(143)-Gly(160) followed by the removal of a Val(161)-Ala(162) dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln(151)-Gly(160) peptide and completed processing at Ser(145) or Gly(143) at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg(163) at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser(145) to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser(145) as well as Asp(146) (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614-11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna.     

Human cathepsin B1. Inhibition by α2-macroglobulin and other serum proteins

1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as alpha(2)-macroglobulin. 3. alpha(2)-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against alpha-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of alpha(2)-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ;cathepsin B' by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.     

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.     

Identification of cathepsin B,D and H in the larval midgut of colorado potato beetle,Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-Ala-Arg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

Membrane-associated cathepsin L: a role in metastasis of melanomas

Subcellular distribution of cathepsin L, the major protein released by transformed or ras transfected fibroblasts, was examined in murine liver, murine B16 amelanotic melanoma and human A2058 melanoma after sequential differential and Percoll density gradient centrifugation. In both murine and human melanomas, cathepsin L activity was found to be enriched in plasma membrane fractions; cathepsin L in these fractions was in both native and acid activatable forms. Plasma membrane fractions from B16 melanoma subpopulations of "low" and "high" metastatic potential were assayed for activity of cathepsin L and of heat stable endogenous inhibitors. The relative specific activity of cathepsin L was 7-fold greater in the subpopulation of "high" metastatic potential, whereas cysteine proteinase inhibitory activity was 5-fold less. Since cathepsin L can degrade intact basement membrane, this membrane-associated cathepsin L may well contribute to metastatic spread of melanomas.     

Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro

Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin B neutralizing monoclonal antibody (Mab) 2A2, together with other general and specific cysteine proteinase inhibitors. We showed that the degradation of extracellular matrix by living MCF-10A neoT cells was predominantly intracellular, as imaged by confocal assays using quenched fluorescent substrate DQ-collagen IV. CA-074, a membrane-impermeable cathepsin B-selective inhibitor and its membrane-permeable analogue CA-074Me showed similar inhibition of invasion at 10 microM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 microM by 42.7%. Tumor cells may internalize monoclonal antibody; therefore, 2A2 Mab could impair both the intracellular and the extracellular fractions of cathepsin B activity. However, both 2A2 Mab and cathepsin B-selective inhibitors were less potent than the general cysteine proteinase inhibitors chicken cystatin and E-64, indicating that other cysteine proteinases, presumably cathepsin L, are involved in invasion. Our results show that intracellular and extracellular cathepsin B activity contribute to in vitro invasion of MCF-10A neoT cells and suggest that inhibitors capable of impairing both fractions have a potential as new anticancer drugs.