Complex Formation of Human Proelastases with Procarboxypeptidases A1 and A2

András Szabó,Claudia Pilsak,Melinda Bence,Heiko Witt,Miklós Sahin-Tóth

doi:10.1074/jbc.m116.743237

Abstract

The pancreas secretes digestive proenzymes typically in their monomeric form. A notable exception is the ternary complex formed by proproteinase E, chymotrypsinogen C, and procarboxypeptidase A (proCPA) in cattle and other ruminants. In the human and pig pancreas binary complexes of proCPA with proelastases were found. To characterize complex formation among human pancreatic protease zymogens in a systematic manner, we performed binding experiments using recombinant proelastases CELA2A, CELA3A, and CELA3B; chymotrypsinogens CTRB1, CTRB2, CTRC, and CTRL1; and procarboxypeptidases CPA1, CPA2, and CPB1. We found that proCELA3B bound not only to proCPA1 (KD 43 nm) but even more tightly to proCPA2 (KD 18 nm), whereas proCELA2A bound weakly to proCPA1 only (KD 152 nm). Surprisingly, proCELA3A, which shares 92% identity with proCELA3B, did not form stable complexes due to the evolutionary replacement of Ala(241) with Gly. The polymorphic nature of position 241 in both CELA3A (∼4% Ala(241) alleles) and CELA3B (∼2% Gly(241) alleles) points to individual variations in complex formation. The functional effect of complex formation was delayed procarboxypeptidase activation due to increased affinity of the inhibitory activation peptide, whereas proelastase activation was unchanged. We conclude that complex formation among human pancreatic protease zymogens is limited to a subset of proelastases and procarboxypeptidases. Complex formation stabilizes the inhibitory activation peptide of procarboxypeptidases and thereby increases zymogen stability and controls activation.

Highlights

The exocrine pancreas produces digestive enzymes including serine- and metalloproteases secreted as inactive precursors that attain their active form in the duodenum
Renewed interest in digestive protease physiology was spurred by the recognition that mutations in cationic trypsinogen (PRSS1), the pancreatic secretory trypsin inhibitor (SPINK1), chymotrypsin C (CTRC) and CPA1 are strongly associated with chronic pancreatitis in humans [3,4,5,6]
Four genes code for chymotrypsinogens (CTRB1, CTRB2, CTRC, and CTRL1) and five genes for chymotrypsinlike proelastases (CELA1, CELA2A, CELA2B, CELA3A, and CELA3B)

Summary

Introduction

The exocrine pancreas produces digestive enzymes including serine- and metalloproteases secreted as inactive precursors (zymogens) that attain their active form in the duodenum. Information obtained from genome and pancreatic transcriptome sequencing allowed correct annotation of the full complement of digestive protease isoforms and opened up avenues for investigations using genetically-defined recombinant enzymes These studies, in turn, led to novel insight into digestive protease function such as the central role of CTRC in the regulation of human trypsinogen [7,8,9] and procarboxypeptidase activation [2], the role of mesotrypsin (PRSS3) in degrading trypsin inhibitors [10, 11], and the significance of mutation-induced digestive protease misfolding in acinar cell stress and chronic pancreatitis [6, 12, 13]. The objective of the present study was to perform a comprehensive analysis of complex formation among human pancreatic protease zymogens using recombinant proenzymes and to characterize the binding affinities and the functional significance of the complexes

Objectives

Results

Conclusion