Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.

Immunoreactivity of sera to a peptide derived from the serotonin 5-HT1A receptor in a group of children with developmental disorders: possible role in non-autistic epilepsy.

The presence of autoantibodies against the serotoninergic 5-HT1A receptor has been reported in serum from an autistic child using radioligand binding studies. It is now well established that, in cardiovascular diseases with an autoimmune component, patients present in their sera autoantibodies directed against the second extracellular loop of some G-protein coupled membrane receptors. We thus investigated by an enzyme-immunoassay method the presence of anti-5-HT1A receptor antibodies in sera of children with developmental disorders using synthetic peptides corresponding to the first and the second extracellular loops of this receptor. The population of children with developmental disorders was divided in autistic children with or without EEG abnormalities, and in non-autistic children with or without EEG abnormalities. We found that 6 out of 10 sera of non-autistic children with an abnormal EEG recognized the second extracellular loop of the 5-HT1A receptor. This is significantly higher than the other groups of children with developmental disorders or a healthy control group. These observations support the existence of an autoimmune component in epilepsy.

Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom.

Cobra cystatin, a new cysteine-proteinase inhibitor of the cystatin superfamily, was isolated from the venom of the Taiwan cobra (Naja naja atra) by affinity chromatography on S-carboxymethylpapain-Sepharose and reverse-phase chromatography. The venom contained two forms of the inhibitor, one of 11870 Da and the other of 12095 Da, as determined by MS, and pI values of 6.2 and 6.1. Cobra cystatin strongly inhibits cysteine proteinases of the papain family, but not calpain. Papain, cathepsin L, cathepsin B and cathepsin S are inhibited with Ki values of 0.19, 0.1, 2.5 and 1.2 nM respectively. The amino acid sequence of cobra cystatin shows that it is a Type 2 cystatin. The amino acid sequence is 73% identical with that of the cystatin in African-puff-adder (Bitis arietans) venom, with which it shares a unique six-residue insertion in a region opposite the reactive inhibitory site. Cobra cystatin is 25-42% identical with other Type 2 cystatins, the most closely related being the recently described human cystatin M, which also has a similar five-residue insertion starting at position 76 (chicken cystatin numbering). A molecular phylogenetic tree of 16 representative members of Family 2 cystatins was constructed by parsimony analysis; it suggests that snake cystatins, together with Tachypleus tridentatus (Japanese horseshoe crab) cystatin and human cystatin M, form a new subfamily within cystatin Family 2.

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

Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p-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-Cys-Gly-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 (Ki = 2 microM) 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 Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu-41p 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.

Production of anti-peptide antibodies directed against the first and the second extracellular loop of the human serotonin 5-HT1A receptor.

The second extracellular loop of the beta-adrenergic and muscarinic acetylcholine receptors was shown to be an autoimmune target for antibodies in several autoimmune diseases. These autoantibodies and the antibodies induced against synthetic peptides corresponding to this loop have pharmacological and physiological properties upon receptor recognition which could explain their pathophysiological role. We here describe the immune properties of the first and second extracellular loops of another G protein-coupled receptor, the serotonin 5-HT1A receptor. The injection in rabbits of the free peptides Y16L and G21G corresponding to the first and second extracellular loops respectively induced anti-peptide antibodies with high titer, demonstrating the presence of a T-cell epitope on each peptide. Interestingly, in contrast to the G21G peptide that induced only anti-G21G antibodies (Ab-2 antibodies), the Y16L peptide induced two populations of antibodies. One recognized only the Y16L peptide (Ab-1 antibodies), the other recognized both peptides (Ab-12 antibodies). This reflects the presence on the two peptides of at least two B-cell epitopes. The fact that the G21G peptide induces only one antibody population might indicate that it possesses one immunodominant epitope involved in the Ab-2 antibody production and one cryptic epitope involved in the cross-reaction with the anti-Y16L antibodies. But only Ab-2 antibodies were able to recognize specifically the human protein receptor expressed in E coli in immunoblot.

A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.

We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate constant kcat/Km of 3.1 10(7) M-1S-1. It is also efficiently hydrolyzed by cathepsin L, although the kcat/Km for this proteinase is about 60-fold lower than that for papain. This change is due to a decrease in kcat, the Km's are almost identical. This allows clear functional discrimination between these two proteinases, and may lead to the development of selective inhibitors for individual cysteine proteinases. Unlike most commonly used papain substrates, Abz-QVVAGA-EDDnp is not hydrolyzed by trypsin. The papain cleavage site was identified as the A-G bond by N-terminal amino acid sequencing. The use of sensitive and specific substrates such as the one described here will prove invaluable for investigating cysteine proteinase activities in parasite infections. The close interaction between papain or cathepsin L with Abz-QVVAGA-EDDnp is compared to that with cystatin inhibitors, which all include a QxVxG consensus segment in their structure.

An immunochemical approach to investigating the mechanism of inhibition of cysteine proteinases by members of the cystatin superfamily.

Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.