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

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme 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 physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stöcker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys.337, 300–307]. To localize and follow the processing of pro‐astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro‐astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro‐astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro‐astacin, present in the cardiac stomach. We conclude from these 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 investigate which of the two endopeptidases found in the digestive tract of crayfish, 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 that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position −1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino‐acid residues in this position, in these cases also astacin‐specific cleavage would be possible.