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 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.

scholarly journals L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

1982 ◽  
Vol 201 (1) ◽  
pp. 189-198 ◽  
Author(s):  
A J Barrett ◽  
A A Kembhavi ◽  
M A Brown ◽  
H Kirschke ◽  
C G Knight ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Competitive Inhibitor ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Leucocyte Elastase ◽  
Structural Analogues ◽  
Stoichiometric Reaction

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

scholarly journals The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues

1989 ◽  
Vol 263 (3) ◽  
pp. 945-949 ◽  
Author(s):  
R W Mason ◽  
L T Bartholomew ◽  
B S Hardwick
Keyword(s):  
Tissue Distribution ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Molecular Size ◽  
Cathepsin S ◽  
Human Tissues ◽  
Cysteine Proteinases ◽  
Additional Protein ◽  
Human Cathepsin ◽  
Kidney Liver

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.

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.

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 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 The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture

1992 ◽  
Vol 283 (2) ◽  
pp. 461-465 ◽  
Author(s):  
B M Cullen ◽  
I M Halliday ◽  
G Kay ◽  
J Nelson ◽  
B Walker
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
Cell Lines ◽  
Cathepsin B ◽  
Breast Tumour ◽  
Tumour Cells ◽  
Affinity Label ◽  
Cells In Culture ◽  
Er Positive ◽  
Breast Tumour Cells

In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.

scholarly journals Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

1998 ◽  
Vol 330 (2) ◽  
pp. 833-838 ◽  
Author(s):  
W. Robert MASON ◽  
Katia SOL-CHURCH ◽  
Magnus ABRAHAMSON
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Terminal Segment ◽  
Cathepsin S ◽  
Amino Acid Substitutions ◽  
Cysteine Proteinases ◽  
Aromatic Residues ◽  
Human Cystatin C

We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases. Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli. It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes. All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues. The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H. Cathepsin L showed a preference for larger aromatic residues in S2. In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant. The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively). The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant. In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM. Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.

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