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J. Biol. Chem., Vol. 280, Issue 33, 29645-29652, August 19, 2005

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The Tetra-aspartate Motif in the Activation Peptide of Human Cationic Trypsinogen Is Essential for Autoactivation Control but Not for Enteropeptidase Recognition^*

Zsófia Nemoda and Miklós Sahin-Tóth

From the Department of Molecular and Cell Biology, Boston University, Goldman School of Dental Medicine, Boston, Massachusetts 02118

The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp^19-22 in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp^19-22 had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala^19-22 trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp^19-22 motif was critical for autoactivation control. Thus, single Ala mutations of Asp¹⁹, Asp²⁰ and Asp²¹ resulted in 2-3-fold increased autoactivation, whereas the Asp²² Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala^19-21 and tetra-Ala^19-22 mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp²¹⁸, which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp²¹⁸ in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp^19-22 motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp²¹⁸, which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.

Received for publication, May 24, 2005 , and in revised form, June 20, 2005.

^* This work was supported by National Institutes of Health Grant DK058088 (to M. S.-T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 715 Albany St., Evans-433; Boston, MA 02118. Tel.: 617-414-1070; Fax: 617-414-1041; E-mail: miklos{at}bu.edu.

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