Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.     

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase

A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle.

Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.     

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.     

Quantitative study of the binding of cysteine proteinases to basement membranes.

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactivated by E64 was used for Kd determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, Kd, and the number of substrate sites, n, values of which were in the range of 10(-7) M and 10(12), respectively. Such results would be of significant interest for the understanding of the biological role of cysteine proteinases in tumour invasion and other types of tissue remodeling.     

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.     

A catalytically active high-Mr form of human cathepsin B from sputum.

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.     

Role of cathepsin B and L in anti-glomerular basement membrane nephritis in rats

We have examined the potential role of the cysteine proteinases, cathepsin B and L, in renal tubular protein degradation and increased permeability of the glomerular basement membrane (GBM) which occurs in a neutrophil- and complement-independent model of anti-GBM antibody disease. The specific activity of cathepsin L, but not cathepsin B, was significantly increased (157%, p greater than 0.01) in cortical homogenates (85-90% tubules) prepared from anti-GBM-treated rats compared to saline-treated controls. Using highly purified cathepsin B and L, we documented the ability of these proteinases to degrade albumin in vitro (Km 5.92 and 0.22 microM for B and L, respectively). In two separate studies, treatment of rats with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, (E-64), a specific and irreversible inhibitor of cysteine proteinases, significantly reduced proteinuria (-45 and -41%, p less than 0.01) in the 24-hour period following injection of the anti-GBM IgG. Taken together, these data suggest an important role for cysteine proteinases in the increased tubular protein degradation which occurs in response to increased filtered protein loads and in the increased GBM permeability (proteinuria) characteristic of glomerular disease.     

Cystatin-like cysteine proteinase inhibitors from human liver.

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].     

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.     

Cathepsin D inactivates cysteine proteinase inhibitors, cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.     

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzedAbz-QVVAGA-EDDnp andAbz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10⁷M^–1 sec^–1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10⁵ M^–1 sec^–1 range due to lowerk _cat values, with theK _m 's being identical to those with papain. OnlyAbz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN₂ was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k ₊₀= 1,100,000 M^–1 sec^–1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN₂. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.     

Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases

The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.     

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1alpha synthase (ST6GalNAc V)(1)

Cystatin C with the 11 N-terminal amino acids truncated shows a much lower affinity for cysteine proteinases than the intact inhibitor. Such truncation of cystatin C is recorded after action of glycyl endopeptidase and cathepsin L. Incubation of cystatin C with papain, cathepsin B or cathepsin H led to no changes in the cystatin C molecule. Isoelectric focusing of the cathepsin L and cystatin C mixture showed the formation of two new bands. One of them appeared whether E-64 or PMSF was added or not, evidently representing a cystatin C/cathepsin L complex. The other band is the truncated cystatin C molecule. N-terminal sequencing after separation by HPLC showed that cystatin C is cleaved by cathepsin L at the Gly11-Gly12 bond. The action of cathepsin L on cystatin C may be explained by the cleavage of the scissile bond in an inappropriate complex.     

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.     

Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin

The comparison of the amino acid sequences of 5 cysteine proteinases: papain, actinidin, rat cathepsins B and H and chicken cathepsin L, demonstrates a striking homology among their sequences. The N-terminal region (residues 1-70 in papain) and C-terminal region (residues 118-212 in papain) display the highest sequence homologies, whereas the lowest sequence homologies are observed in the middle region (residues 71-117 in papain); a segment where most insertions/deletions are observed. The highest sequence homology is observed between rat cathepsin H and chicken cathepsin L. As shown by X-ray studies, papain and actinidin have a clearly defined double domain structure. Each domain contains a core of non-polar side chains, which are retained in cathepsins B, H and L, except for the non-polar residue 203 of the core which is replaced by glutamic acid in cathepsin B. The percentage and the location of alpha-helix and beta-sheets of cathepsins B, H and L, assessed using the methods of Garnier et al. (1978, J. Mol. Biol. 120, 97-120) and Chou and Fasman (1974, Biochemistry 13, 222-245), show that the main ordered structures in papain and actinidin are probably retained in cathepsins B, H and L. The differences observed occur essentially in the middle region, a place where sequences display the lowest homologies and which is far removed from the active site.