A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation

Abstract

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

An expression profile of human pancreatic islet mRNAs by Serial Analysis of Gene Expression (SAGE)

“…This stress might be reflected in the current libraries through the very high expression level of several factors such as reg-1α and -1β, Hsp 70, and clusterin. Reg-1α is known to be expressed in both exocrine cells [16] and regenerating islets [15,28,29,30]. The abundance of reg in pancreatic juice (10 to 14% of total protein) suggests that it plays an important role in exocrine pancreatic function.…”

“…In contrast, in the gradient purified library that was further hand-selected, the relative exocrine content appeared to be diminished (compare Tables 2 and 3). Notably, many stress response related transcripts like reg-1α and β, clusterin or heat-shock proteins were also present at high levels in the different libraries, perhaps reflecting stress the cells were subjected to during the preparation of the tissues [15,16,17].…”

Small-Interference RNA Gene Knockdown of Pancreatitis-Associated Proteins in Rat Acute Pancreatitis

“…Anti-PAP antibody is species specific and has minimal overlap with other Reg isoforms (16). PAP protein size ranges from 13-18Kd and is dependent on PAP cleavage, post translational modification and glycosylation (17,18).…”

Human Pancreatitis-associated Protein Forms Fibrillar Aggregates with a Native-like Conformation

“…Both PAP and LIT contain a carbohydrate recognition domain linked to a signal peptide and belong to group VII of the C-type lectin family (28). Rat PAP I and III (an isoform of rat PAP) and LIT can form highly organized fibrillar structures following trypsin cleavage, which removes the N-terminal propeptide and converts the 16-kDa proteins into the 14-kDa mature isoforms (29). However, it is difficult to understand how precipitation properties could serve a useful function in pancreatic physiology.…”