Abstract
Mutation of Arg(117), an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.
Highlights
Ated with several mutations in the PRSS1 gene encoding human cationic trypsinogen (Tg)
Studies used recombinant rat anionic Tg as a model for human cationic Tg [15,16,17,18], but, lately, techniques have been developed that allowed recombinant expression and in vitro refolding of the human enzyme (19 –22). Experiments using these recombinant cationic Tg preparations have addressed four major aspects of Tg/Tr biochemistry, with the following developments: (i) Autocatalytic degradation of Tr was inhibited by mutations R117H [20, 21], R117C [3], and N21T [19], whereas mutation N21I (19 –22) had no such effect. (ii) Autocatalytic activation of Tg was enhanced by all four mutations (N21T, N21I, R117H, R117C) studied so far (3, 19 –22)
Autocatalytic cleavage at Arg117 was first described more than 30 years ago in preparations of bovine cationic Tr [28], and it has been documented in porcine [29], rat (16 –18, 33), and human [19, 34] Tr or Tg since
Summary
Arg117 IS THE REACTIVE SITE OF AN INHIBITORY SURFACE LOOP THAT CONTROLS SPONTANEOUS ZYMOGEN ACTIVATION*. In the absence of Ca2؉, at pH 8.0, the hydrolysis equilibrium (Khyd [ ؍cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mM Ca2؉ reduced the rate of cleavage at Arg117 at least 20-fold, and shifted Khyd to 0.7 These observations indicate that the Arg117-Val118 bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. We found that Tr cleaved the Arg117-Val118 bond and re-synthesized it until an equilibrium was reached between the cleaved and intact Tg forms These properties of Arg117 resemble the reactive site of reversible canonical protease inhibitors [24] and suggest that Arg117 plays an inhibitory role in controlling zymogen activation in the pancreas. Evidence to support this notion is presented here, and a model describing the dual role of Tr in protecting against pathological Tg activation is discussed
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