scholarly journals Collagenolytic cysteine proteinases of bone tissue. Cathepsin B, (pro)cathepsin L and a cathepsin L-like 70 kDa proteinase

1991 ◽  
Vol 279 (1) ◽  
pp. 167-174 ◽  
Author(s):  
J M Delaissé ◽  
P Ledent ◽  
G Vaes
Keyword(s):  
Molecular Mass ◽  
Bone Tissue ◽  
Cathepsin B ◽  
Hydrophobic Interactions ◽  
Relative Rate ◽  
Cathepsin L ◽  
Gel Filtration ◽  
Cysteine Proteinases ◽  
Western Blots ◽  
Mouse Muscle

The aim of the work was to identify and characterize the cysteine proteinases of bone tissue, as these enzymes appear necessary for bone resorption. Three cysteine-dependent proteolytic activities were separated from a homogenate of mouse calvaria by a fractionation procedure involving (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The first two are typical cathepsins B and L with respect to (1) their reactivity with anti-(cathepsin B) and anti-(cathepsin L) antibodies respectively, (2) their relative rate constants for inhibition by benzyloxycarbonyl-Phe-Phe-CHN2 and L-3-carboxy-trans-2,3-epoxypropionyl-L-leucylamido-(4-guanid ino)butane and (3) their enzymic properties, such as the higher activities of cathepsin L against collagen and gelatin as compared with cathepsin B, and the fact that benzyloxycarbonyl-Arg-Arg 4-methoxy-2-naphthylamide is hydrolysed only by cathepsin B. Cathepsin L was mainly recovered in its precursor form, as indicated by its apparent 40 kDa molecular mass and its relative stability at pH 7.2. The third enzyme is a cathepsin L-like proteinase with an apparent molecular mass of 70 kDa. It is immunoprecipitated by anti-(cathepsin L) antibodies, and appears as the 25 kDa band of mature cathepsin L in Western blots. It further resembles (pro)cathepsin L with regard to its activities against synthetic substrates and proteins such as collagen, and with regard to its response to various inhibitors. However, unlike (pro)cathepsin L, it is eluted as a 70 kDa protein on gel filtration (even in the presence of 1% Brij or 1 M-NaCl), it is stable at pH values as high as 9, and it exhibits stronger affinity for phenyl-Sepharose. It might thus result from a strong complex between mature cathepsin L and another entity that confers stability at alkaline pH and favours hydrophobic interactions. This 70 kDa activity was also detected in mouse muscle and long bones of Ca(2+)-deficient chicks but not in mouse liver, spleen or kidney.

The cysteine proteinases of Schistosoma mansoni cercariae

Parasitology ◽  
1997 ◽  
Vol 114 (2) ◽  
pp. 105-112 ◽  
Author(s):  
J. P. DALTON ◽  
K. A. CLOUGH ◽  
M. K. JONES ◽  
P. J. BRINDLEY
Keyword(s):  
Schistosoma Mansoni ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Skin Penetration ◽  
Scanning Electron Micrographs ◽  
Cysteine Proteinases ◽  
Similar Function ◽  
Substrate Preferences ◽  
Serine Proteinase Activity

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

Purification and properties of different isoforms of bovine cathepsin B

1990 ◽  
Vol 68 (4) ◽  
pp. 822-826 ◽  
Author(s):  
Christiane Deval ◽  
Daniel Bechet ◽  
Alain Obled ◽  
Marc Ferrara
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Bovine Liver ◽  
Relative Mass ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Different Populations ◽  
Rapid Purification

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30 000) and cleaved (Mr 25 000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 μM and 52 s−1, and 125 μM and 75 s−1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.Key words: protein/enzyme purification, cathepsin B, isoforms, lysosomes.

scholarly journals The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor

1989 ◽  
Vol 257 (1) ◽  
pp. 125-129 ◽  
Author(s):  
R W Mason ◽  
D Wilcox ◽  
P Wikstrom ◽  
E N Shaw
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Active Form ◽  
Culture Conditions ◽  
Secreted Protein ◽  
Cysteine Proteinases ◽  
Tumour Invasion ◽  
Specific Antibodies ◽  
Mouse Fibroblasts

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.

scholarly journals Quantification of cathepsins B and L in cells

1998 ◽  
Vol 332 (2) ◽  
pp. 499-505 ◽  
Author(s):  
Ruye XING ◽  
Adele K. ADDINGTON ◽  
Robert W. MASON
Keyword(s):  
Active Site ◽  
Total Protein ◽  
Cathepsin B ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cathepsin L ◽  
Breast Tumour ◽  
Cathepsin S ◽  
Cysteine Proteinases ◽  
Breast Tumour Cells

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.

scholarly journals A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen

1988 ◽  
Vol 256 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R A Maciewicz ◽  
D J Etherington
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cross Reactivity ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Collagenolytic Activity ◽  
Cysteine Proteinases ◽  
Isoelectric Points ◽  
Multiple Charge

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.

scholarly journals The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B

1988 ◽  
Vol 253 (3) ◽  
pp. 751-758 ◽  
Author(s):  
C Crawford ◽  
R W Mason ◽  
P Wikstrom ◽  
E Shaw
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Inhibitory Effects ◽  
Enzyme Functions ◽  
Calpain Ii

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.

scholarly journals Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi

1996 ◽  
Vol 318 (2) ◽  
pp. 395-399 ◽  
Author(s):  
Gilles LALMANACH ◽  
Roger MAYER ◽  
Carole SERVEAU ◽  
Julio SCHARFSTEIN ◽  
Francis GAUTHIER
Keyword(s):  
Trypanosoma Cruzi ◽  
Cathepsin B ◽  
Active Sites ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Irreversible Inhibitors ◽  
Human Cystatin C ◽  
Spacer Arm ◽  
Biotin Labelling

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Effect of Cysteine Proteinase Inhibitors on Murine B16 Melanoma Cell Invasion in vitro

2002 ◽  
Vol 383 (5) ◽  
pp. 839-842 ◽  
Author(s):  
Natasa Sever ◽  
Metka Filipic ◽  
Joze Brzin ◽  
Tamara T. Lah
Keyword(s):  
Cell Invasion ◽  
Cathepsin B ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Cysteine Proteinase Inhibitors

Abstract Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

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