Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (K_i) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human neutrophil elastase inhibitors described in a mollusk.