Background: Reg and PAP are homologous, endogenous proteins differentially expressed in the pancreas. We recently showed that PAP2 is protective in pancreatitis, and it may exert its effect by activating macrophages. Since RegI is constituitively expressed and PAP2 is induced only during pancreatitis, we postulated that they would have differential effects on macrophages. We also postulated that the highly conserved C-terminus, previously identified as critical for this activity, would be a bioactive fragment.
Methods: Rat macrophages (NR8383 alveolar cell line) were cultured with recombinant rat RegI or PAP2 proteins created in our lab, and isolated by affinity chromatography, a commercial PAP2 protein isolated by NH4 precipitation, or a synthetic 30 amino acid C-terminus peptide (20-10000 ng/ml) for 24 hr. Media was evaluated for TNF expression via ELISA. Controls consisted of cells cultured with vehicle alone. Significance was set at p < 0.05 (Student’s t-test).
Results: See Figure. Macrophages cultured in the presence of our laboratory’s recombinant rat RegI and PAP2 resulted in increased expression of TNFa when compared with controls (p<0.05). But, PAP2 was 2 log-orders more potent. The highly conserved C-terminal peptide showed no activity. Importantly, commercially purchased PAP2 isolated by ammonium sulfate precipitation was inactive; its activity returned only after chemical refolding using serial dialysis in urea.
Conclusions: Despite their structural similarities, PAP2 is a much more potent macrophage stimulant than RegI. Since PAP2 is induced during acute pancreatitis, it is likely to be the more important signal for macrophage activation in the disease. A conserved peptide sequence is inactive, intact conformational structure is critical for its activity, and standard isolation techniques can render the protein non-functional.