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J. Biol. Chem., Vol. 283, Issue 20, 13943-13951, May 16, 2008

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Guinea Pig Chymase Is Leucine-specific

A NOVEL EXAMPLE OF FUNCTIONAL PLASTICITY IN THE CHYMASE/GRANZYME FAMILY OF SERINE PEPTIDASES^*

George H. Caughey^¶1, Jeremy Beauchamp^||, Daniel Schlatter^||, Wilfred W. Raymond, Neil N. Trivedi, David Banner^||, Harald Mauser^||, and Jürgen Fingerle^||

From the Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143, the ^¶Veterans Health Research Institute and Veterans Affairs Medical Center, San Francisco, California 94121, and ^||Preclinical Pharma Research Basel, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig -chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe⁸ to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by ₁-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster -chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala²¹⁶ side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.

Received for publication, December 26, 2007 , and in revised form, March 13, 2008.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AM851020.

^* This work was supported, in whole or in part, by National Institute of Health Grants HL024136 (to G. H. C. and W. W. R.) and T32 07185 (to N. N. 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.

¹ To whom correspondence should be addressed: Veterans Affairs Medical Center 111-D, 4150 Clement St., San Francisco, CA 94121. Fax: 415-387-3568; E-mail: george.caughey{at}ucsf.edu.

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