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Acknowledgments We thank “Servicio de secuenciación de DNA” and “Servicio de Química de proteínas y Síntesis de oligonucleótidos” at CIB-CSIC, Madrid, and “Unidad de Proteómica” at CNIC-ISCIII, Madrid for the kind assistance of their staff. This work was supported by grants from European Commission (QLRT-2001-01969), and Ministerio de Educación y Ciencia (CICYT BIO2003-03428)., The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optim, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub more...