Structural requirements of 5-hydroxytryptamine-moduline analogues to interact with the 5-hydroxytryptamine1B receptor.

5-Hydroxytryptamine-moduline is an endogenous cerebral tetrapeptide that regulates the activity of 5-hydroxytryptamine1B receptors. Direct binding of 5-[3H]hydroxytryptamine-moduline on rat brain homogenate evidenced the existence of two interacting sites for the peptide, very likely corresponding to different conformations of the 5-hydroxytryptamine1B receptor: The peptide first binds to a low-affinity state of the receptor (pIC50 = 7.68+/-0.14) and then induces (or stabilizes) a high-affinity complex (pIC50 = 11.62+/-0.18). This work focuses on the ability of 5-hydroxytryptamine-moduline analogues to recognize the high- and low-affinity sites for 5-hydroxytryptamine-moduline. The results obtained show that the two conformers of the 5-hydroxytryptamine1B receptor have similar but not identical binding pockets for 5-hydroxytryptamine-moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low-affinity site than the high-affinity one, which only needs a short hydrophobic amino acid in position 2.

Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides.

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7-8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1 +/- 3.6 microM and a Vmax of 5.5 micromol min(-1) l(-1) per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726-735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

A complementarity-determining region peptide of an anti-desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence.

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity-determining region of the F12 heavy chain (VH-CDR3) was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen-combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH-CDR3 inhibited its binding to target antigens while VH-CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self-antigen binding mechanism for some autoantibodies.

Chemical synthesis, structural and functional characterisation of noxiustoxin, a powerful blocker of lymphocyte voltage-dependent K+ channels.

Two forms of the Centrudoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K+ channels with 2 nM affinity, but does not affect the Ca(2+)-activated K+ channels. This toxin, because of its high affinity and specificity for voltage-gated K+ channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.