Abstract
Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.
Highlights
Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors
Because human trypsins are posttranslationally sulfated in the autolysis loop on Tyr-154 [21,22,23,24], we studied the effect of this modification on mesotrypsin activity and its regulation by chymotrypsin C (CTRC)
Human trypsinogens are sulfated on Tyr-154, which forms part of the S2Ј substrate-binding subsite in trypsin (Fig. 1B) [21,22,23,24]
Summary
When mesotrypsinogen was incubated with 5 nM CTRC in the absence of calcium, multiple bands migrating in the 10 –20kDa range on reducing SDS-PAGE were generated (Fig. 1A). Time course of enteropeptidase-mediated activation was followed, nonsulfated and sulfated L81A-mesotrypsinogen developed high trypsin activity, whereas activity of the CTRCcleaved forms was markedly reduced (Fig. 3). CTRC-mediated cleavage of the autolysis loop in L81A-mesotrypsin increased Km values by about an order of magnitude, whereas kcat values were relatively unaffected. To examine catalytic activity on a larger protein substrate, we digested bovine -casein with cleaved and uncleaved L81Amesotrypsin, using both nonsulfated and sulfated forms (Fig. 4). To examine the effect of CTRC-mediated cleavage of the autolysis loop on inhibitor binding, we measured binding of SBTI to sulfated L81A-mesotrypsin. We studied the digestion of SBTI by intact and CTRC-cleaved L81A-mesotrypsin (nonsulfated and sulfated) using reducing SDS-PAGE (Fig. 6). When cleaved in the autolysis loop by CTRC, both nonsulfated and sulfated L81A-mesotrypsin proved to be essentially inactive in SBTI digestion and generated only very faint cleavage products
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