Cysteine‐proteinase‐inhibiting function of T kininogen and of its proteolytic fragments

Moreau, Thierry; Esnard, F.; Gutman, N; Degand, Pierre; Gauthier, Francis

doi:10.1111/j.1432-1033.1988.tb13983.x

Cited by 36 publications

(17 citation statements)

References 31 publications

(5 reference statements)

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“…Rat liver cathepsin B was purified as previously described [17]. Recombinant human cathepsin B was a gift from Dr. John S. Mort (McGill University, Montreal, Canada).…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

et al. 1996

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Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues lp-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-CysGly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Kt = 2 ltM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Kl values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PBll at the Lys-40p-Leu41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.

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“…Rat liver cathepsin B was purified as previously described [17]. Recombinant human cathepsin B was a gift from Dr. John S. Mort (McGill University, Montreal, Canada).…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

et al. 1996

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“…The recombinant catalytic domain of cruzipain was a gift from Dr. James McKerrow (University of California, San Francisco, CA). Cathepsins B and L were purified from rat liver (32). The activation buffers for enzyme assays were 0.1 M phosphate buffer, pH 6.0, containing 6 mM DTT and 2 mM EDTA for congopain, and 0.1 M phosphate buffer, pH 6.0, containing 10 mM DTT for cruzipain, 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA, 2 mM DTT for cathepsins B and L. Titration of the active site was made using E64 (33).…”

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confidence: 99%

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

Lalmanach

Lecaille

Chagas

et al. 1998

Journal of Biological Chemistry

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The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in K i value was obtained upon Nterminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.Cysteine proteinases of the papain superfamily have very similar sequences and a common catalytic mechanism; they are found in bacteria, protozoa, plants, and mammals (1, 2). These enzymes play a critical role in the life cycle of protozoan parasites, in host invasion and alteration of the host immune response (3, 4). Parasite cysteine proteinases from the genus Trypanosoma and Plasmodium, have been reported as putative targets for chemotherapeutic inhibitors (5, 6). The main drawback of this strategy is that cysteine proteinases have a broad substrate specificity, which makes it difficult to develop inhibitors that target individual proteinases.Congopain is a cathepsin L-like cysteine proteinase of Trypanosoma congolense, and cruzipain is the equivalent from Trypanosoma cruzi. These protozoan parasites cause bovine trypanosomiase in Africa and Chagas disease in South America respectively (3, 4). Cruzipain (also called cruzain) is a highly mannosylated glycoprotein and an immunodominant antigen (GP57/51) that is among the best characterized of parasitic cysteine proteinases (7-11). The three-dimensional structure of its recombinant central catalytic domain (215 residues) is very similar to those of papain and cathepsin L (11, 12). Congopain differs only slightly from cruzipain in its enzymatic specifici...

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“…Rat mast cell chymase was purified by the procedure of Kido et al [27] and rat liver cathepsin L as reported in [23]. Papain was purchased from Boehringer Mannheim.…”

Section: Enzymesmentioning

confidence: 99%

“…Plasma was obtained from turpentine-injected Wistar rats and used to prepare thiostatin as described previously [23].…”

Section: Biological Materialsmentioning

confidence: 99%

Discrimination between rat thiostatin (T‐kininogen) and one of its cystatin‐like inhibitory fragments by a monoclonal antibody, and localization of the epitope

Lalmanach

Adam

Moreau

et al. 1991

European Journal of Biochemistry

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A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in TI. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.

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Cysteine‐proteinase‐inhibiting function of T kininogen and of its proteolytic fragments

Cited by 36 publications

References 31 publications

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

Discrimination between rat thiostatin (T‐kininogen) and one of its cystatin‐like inhibitory fragments by a monoclonal antibody, and localization of the epitope

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