European Journal of PROTISTOLOGY European Journal of Protistology 44 (2008) 103–113 Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae Andre´ Luis Souza dos Santos a,Ã , Rosangela Maria de Arau´jo Soares a , Celuta Sales Alviano a , Lucimar Ferreira Kneipp b a Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´es, Universidade Federal do Rio de Janeiro, Cidade Universita´ria, Ilha do Fundao, 21941-902 Rio de Janeiro, RJ, Brazil b Departamento de Micologia, Instituto Oswaldo Cruz–Fundac ¸a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil Received 24 May 2007; received in revised form 25 July 2007; accepted 23 August 2007 Abstract Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite–host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS–PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc- chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae. Crown Copyright r 2007 Published by Elsevier GmbH. All rights reserved. Keywords: Cellular proteolytic enzymes; Metallopeptidases; Trypanosomatidae Introduction The family Trypanosomatidae (order Kinetoplastida) is composed exclusively of parasitic organisms with a single flagellum and a kinetoplast (Vickerman 1994). Trypanosomatids parasitize all classes of vertebrates as well as some invertebrates, preferentially insects from the orders Diptera and Hemiptera, and also plants. Invertebrates act as hosts of monogenetic parasites, such ARTICLE IN PRESS www.elsevier.de/ejop 0932-4739/$ - see front matter Crown Copyright r 2007 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2007.08.006 Ã Corresponding author. Laborato´rio de Estudos Integrados em Bioquı´mica Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´es (IMPPG), Bloco I- subsolo, Centro de Cieˆncias da Sau´ de (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Avenida Carlos Chagas Filho, 373, Cidade Universita´ria, Rio de Janeiro, RJ 21941-902, Brazil. Tel.: +5521 2562 6740; fax: +5521 2560 8344. E-mail address: andre@micro.ufrj.br (A.L.S. Santos).