The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

Download Full-text

Action of human liver cathepsin B on the oxidized insulin B chain

Biochemical Journal ◽

10.1042/bj2130467 ◽

1983 ◽

Vol 213 (2) ◽

pp. 467-471 ◽

Cited By ~ 34

Author(s):

M J McKay ◽

M K Offermann ◽

A J Barrett ◽

J S Bond

Keyword(s):

Amino Acid ◽

Cathepsin B ◽

Human Liver ◽

Degradation Products ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Amino Acid Content ◽

Peptide Bond ◽

Cleavage Sites ◽

Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

Download Full-text

Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8

Biochemical Journal ◽

10.1042/bj3500563 ◽

2000 ◽

Vol 350 (2) ◽

pp. 563-568 ◽

Cited By ~ 135

Author(s):

Henning R. STENNICKE ◽

Martin RENATUS ◽

Morten MELDAL ◽

Guy S. SALVESEN

Keyword(s):

Peptide Bond ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Peptide Substrates ◽

Peptide Cleavage ◽

Fluorescent Peptide ◽

General Order ◽

Caspase Family ◽

General Preference ◽

Individual Profiles

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P4, P1 and P1´ subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S4 subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S1 and S1´ subsites that flank the scissile peptide bond. The S1 subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P1´ position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P1´ subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Download Full-text

Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor

Biological Chemistry ◽

10.1515/bc.2010.049 ◽

2010 ◽

Vol 391 (5) ◽

pp. 561-570 ◽

Cited By ~ 5

Author(s):

Larissa P. Coppini ◽

Nilana M.T. Barros ◽

Marcela Oliveira ◽

Izaura Y. Hirata ◽

Marcio F.M. Alves ◽

...

Keyword(s):

Cathepsin L ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Resonance Energy ◽

Peptide Bond ◽

Fibrin Clot ◽

Cathepsin S ◽

Enzyme Specificity ◽

Amino Terminal ◽

Cathepsin V

Abstract Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S3′-P3′ interaction indicates the significance of the extended subsites for enzyme specificity and affinity.

Download Full-text

Cathepsin S The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15)

Biochemical Journal ◽

10.1042/bj2400455 ◽

1986 ◽

Vol 240 (2) ◽

pp. 455-459 ◽

Cited By ~ 52

Author(s):

H Kirschke ◽

I Schmidt ◽

B Wiederanders

Keyword(s):

Rat Liver ◽

Polyclonal Antibody ◽

Lymphoid Tissue ◽

Cysteine Proteinase ◽

Rat Kidney ◽

Cathepsin L ◽

Bovine Liver ◽

Cathepsin S ◽

Irreversible Inhibitor ◽

C 50

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.

Download Full-text

Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity

Biochemical Journal ◽

10.1042/bj20030342 ◽

2003 ◽

Vol 373 (3) ◽

pp. 933-939 ◽

Cited By ~ 21

Author(s):

Jefferson P. HEMERLY ◽

Vitor OLIVEIRA ◽

Elaine DEL NERY ◽

Rory E. MORTY ◽

Norma W. ANDREWS ◽

...

Keyword(s):

Amino Acids ◽

Trypanosoma Cruzi ◽

Trypanosoma Brucei ◽

Peptide Bond ◽

Minimum Size ◽

Amino Acid Residues ◽

Basic Amino Acids ◽

Oligopeptidase B ◽

Work Supports ◽

Micromolar Range

We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1′, S2′ and S3′ subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg–Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg–Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1′ subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1′ resulted in substrates that were hydrolysed with Km values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high kcat and low Km values.

Download Full-text

Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages

Journal of Experimental Medicine ◽

10.1084/jem.20020978 ◽

2003 ◽

Vol 197 (2) ◽

pp. 169-179 ◽

Cited By ~ 79

Author(s):

Courtney Beers ◽

Karen Honey ◽

Susan Fink ◽

Katherine Forbush ◽

Alexander Rudensky

Keyword(s):

Cathepsin L ◽

Specific Inhibitor ◽

Cell Types ◽

Interferon Γ ◽

Cathepsin S ◽

Antigen Presenting Cell ◽

Relative Contribution ◽

Helper Cell Type ◽

Ifn Γ ◽

Antigen Presenting

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mϕs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mϕs. In addition, our studies show that the level of catL activity is significantly decreased in Mϕs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mϕs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mϕs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mϕs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.

Download Full-text

Cleavage of immunoglobulin G by excretory–secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

Parasitology ◽

10.1017/s0031182000076496 ◽

1994 ◽

Vol 109 (5) ◽

pp. 611-621 ◽

Cited By ~ 56

Author(s):

Y. Kong ◽

Y.-B. Chung ◽

S.-Y. Cho ◽

S.-Y. Kang

Keyword(s):

Immunoglobulin G ◽

Cathepsin L ◽

Immunoblot Analysis ◽

Cathepsin S ◽

Terminal Amino Acid ◽

Optimal Activity ◽

Spirometra Mansoni ◽

Terminal Amino ◽

Secretory Products ◽

Ph Range

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of Mr 27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5 M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 × 10−5 M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

Download Full-text

A systematic series of synthetic chromophoric substrates for aspartic proteinases

Biochemical Journal ◽

10.1042/bj2370899 ◽

1986 ◽

Vol 237 (3) ◽

pp. 899-906 ◽

Cited By ~ 136

Author(s):

B M Dunn ◽

M Jimenez ◽

B F Parten ◽

M J Valler ◽

C E Rolph ◽

...

Keyword(s):

Structure Function ◽

Peptide Bond ◽

Water Soluble ◽

Animal Origin ◽

Aspartic Proteinases ◽

Peptide Substrates ◽

Representative Selection ◽

Micro Organisms ◽

Hydrolysis Of ◽

Selection Of

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

Download Full-text

Possible Involvement of Cathepsin L in Processing of Rat Liver Hexokinase to Eliminate Mitochondria-Binding Ability

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a123913 ◽

1992 ◽

Vol 112 (3) ◽

pp. 409-413 ◽

Cited By ~ 2

Author(s):

Hiroshi Okazaki ◽

Chiemi Tanl ◽

Miyuki Ando ◽

Kyoko Ishii ◽

Sadahiko Ishibashi ◽

...

Keyword(s):

Rat Liver ◽

Cathepsin L ◽

Binding Ability

Download Full-text

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.83-91.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 83-91

Author(s):

S Miyazawa ◽

T Osumi ◽

T Hashimoto ◽

K Ohno ◽

S Miura ◽

...

Keyword(s):

Amino Acid ◽

Rat Liver ◽

Coenzyme A ◽

Terminal Region ◽

Proteinase K ◽

Amino Acid Residues ◽

C Terminus ◽

Targeting Signal ◽

Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Download Full-text