Activation of pro-astacin - Immunological and model peptide studies on theprocessing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus

To contribute knowledge of the processing and activation of invertebrate pr oteolytic enzymes, we studied the metalloprotease astacin, a digestive enzy me from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiologi cal reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stocker, W. & Zwilling, R. (1997) Arch. Bio chem. Biophys. 337, 300-307]. To localize and follow the processing of pro- astacin in different parts of the digestive tract, we synthesized two pepti des covering the pro part of pro-astacin and raised antibodies against them . In addition, antiserum against the whole active astacin was produced. Usi ng immunohistochemical investigation, we detected pro-astacin in the F cell s of the hepatopancreas and all the way into the tubular lumen and the coll ecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from th ese studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To inv estigate which of the two endopeptidases found in the digestive tract of cr ayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed th at astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substitu ting glycine for arginine at position -1 of the activation site does not pr event astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-spe cific cleavage would be possible.