Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis

Abstract

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.