MOLECULAR ANALYSIS OF HEREDITARY PANCREATITIS ASSOCIATED PRSS1 MUTATIONS

Background: Hereditary pancreatitis is caused by mutations in the cationic trypsinogen (PRSS1) gene. Although several PRSS1 mutations were reported the molecular mechanisms underlying the pathophysiological processes remain unclear. Many of the identified PRSS1 mutations affect autocleavage sites or sequences involved in the physiological or pathophysiological trypsinogen activation. Here we have applied a newly developed FRET assay to analyse the effects of the reported PRSS1 mutations on the kinetics of PRSS1 peptide digestion by enzymes involved in physiological and pathophysiological trypsin cleavage. Methods: The degradation kinetics of the mutated PRSS1 sequences were measured by a fluorescence resonance energy transfer (FRET) assay. We have designed 9 -14 amino acid peptides containing the PRSS1 mutations A16V, D19A, D22G, K23R, N29I, N29T, R116C, A121T, R122H, R122C and V123M coupled to a DABCYL/EDANS FRET system. The fluorescence was quenched in intact peptides. Cleavage resulted in fluorescence development. Wild type and peptides derived from the mutated sequences were incubated in pH 5.0 or pH 8.0 HEPES buffer in the presence of bovine pancreas trypsin, cathepsin B, enterokinase or pancreas elastase for 30 min. The kinetic of peptide cleavage was measured in a Fluorescence ELISA reader. Results: Peptides containing N29Iand N29T mutations were much more efficiently cleaved by pancreatic elastase than the wild type peptide. Mutations at D19A, D22G and K23R increased the rate of enterokinase digestion, A16V mutation caused a very strong increase. D22G and K23R also resulted in a significantly increased trypsin digestion. As expected R116C and R122C mutations inhibited the digestion of the peptides by trypsin. N29Tmutation induced the cleavage by cathepsin B whereas the mutated A16V and the D22G peptides were not cleaved in contrast to the wild type peptides. Conclusions: With the new FRET assay we have established a fast and reliable method for the analysis of digestion kinetics of mutated PRSS1 sequences. The obtained data sets give new insights in the molecular mechanisms involved in the development of hereditary pancreatitis.