Human cationic trypsinogen -: Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis

Mutation Asn-21 --> Ile in human cationic trypsinogen (Tg-l) has been associated with hereditary pancreatitis. Recent studies with rat anionic Tg (Tg-2) indicated that the analogous Thr-21 --> Ile mutation stabilizes the zymogen against autoactivation, whereas it has no effect on catalytic properties or autolytic stability of trypsin (Sahin-Toth, M. (1999) J. Biol. Chem. 274, 29899-29704). In the present paper, human cationic Tg (Asn-81-Tg) and mutants Asn-21 --> Ile (Ile-21-Tg) and Asn-21 --> Thr (Thr-21-Tg) were expressed in Escherichia coli, and zymogen activation, zymogen degradation, and trypsin autolysis were studied. Enterokinase activated Asn-21-Tg approximately 2-fold better than Ile-21-Tg or Thr-21-Tg, and catalytic parameters of trypsins were comparable. At 37 degrees C, in 5 mM Ca2+, all three trypsins were highly stable. In the absence of Ca2+, Asn-21- and Ile-21-trypsins suffered autolysis in an indistinguishable manner, whereas Thr-21-trypsin exhibited significantly increased stability. In sharp contrast to observations with the rat proenzyme, at pH 8.0, 37 degrees C, autoactivation kinetics of Asn-21-Tg and Ile-21-Tg were identical; however, at pH 5.0, Ile-21-Tg autoactivated at an enhanced rate relative to Asn-a1-Tg. Remarkably, at both pH values, Thr-21-Tg showed markedly higher autoactivation rates than the two other zymogens, Finally, autocatalytic proteolysis of human zymogens was limited to cleavage at Arg-117, and no digestion at Lys-188 was detected. The observations indicate that zymogen stabilization by Ile-21 as observed in rat Tg-2 is not characteristic of human Tg-1, Instead, an increased propensity to autoactivation under acidic conditions might be relevant to the pathomechanism of the Asn-21 --> Ile mutation in hereditary pancreatitis, In the same context, faster autoactivation and increased trypsin stability caused by the Asn-21 --> Thr mutation in human Tg-1 might provide a rationale for the evolutionary divergence from Thr-al found in other mammalian trypsinogens.