Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of t he pancreatic acinar cell, is highly upregulated early in acute pancreatiti s. PAP expression returns to undetectable levels when the pancreas recovers . In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic ston e protein (PSP/reg), which shows strong sequence homology to PAP, is secret ed into pancreatic juice under physiologic and pathologic conditions. PSP/r eg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond. Cleav age results in an:V-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs o r stones from patients with chronic pancreatitis. Rat PAP contains a trypsi n cleavage site at the same position as PSP/reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of r at PAP I, II, and III in a yeast expression system using Pichia pastoris. W e demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis associat ed thread protein (PATP). PATP displays pH-dependent solubility characteris tics very similar to those of PTP.