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J. Biol. Chem., Vol. 280, Issue 12, 10925-10937, March 25, 2005

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Oligopeptidase B from Trypanosoma evansi

A PARASITE PEPTIDASE THAT INACTIVATES ATRIAL NATRIURETIC FACTOR IN THE BLOODSTREAM OF INFECTED HOSTS^*

Rory E. Morty, Roger Pellé¶, István Vadász, Graciela L. Uzcanga||**, Werner Seeger, and José Bubis**

From the Department of Internal Medicine, University of Giessen Medical Centre, Aulweg 123 (Raum 6-11), D-35392 Giessen, Germany, the ^¶International Livestock Research Institute, P. O. Box 30709, Nairobi, Kenya, and the ^**Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of oligopeptidase B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed substrates with basic residues in P₁ (k_cat/K_m for carbobenzyloxy-L-arginyl-L-arginyl-7-amido-4-methylcoumarin, 337 s^–1·µM^–1) and exhibited potent arginyl carboxypeptidase activity (k_cat/K_m for Val-Lys-ArgArg-OH, 231 s^–1·mM^–1). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg³Arg⁴, Arg⁴Ser⁵, Arg¹¹Ile¹², and Arg²⁷Tyr²⁸, thereby abrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg·ml^–1, and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhibitors to cell-free plasma from infected rodents significantly abrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.

Received for publication, September 1, 2004 , and in revised form, January 10, 2005.

^* This work was supported by grants from the Alexander von Humboldt Foundation (to R. E. M.), the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 547: "Kardiopulmonales Gefässsystem" (to R. E. M., I. V., and W. S.), and Fondo Nacional de Ciencia, Tecnología e Innovación Grant G-2000001152 (to J. B.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY546084.

^|| Present address: Dept. de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro s/n, 28040 Madrid, Spain.

To whom correspondence should be addressed. Tel: 49-(641)-994-2303; Fax: 49-(641)-994-2308; E-mail: rory.morty{at}innere.med.unigiessen.de.

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