RT Journal Article
T1 Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification
A1 Díaz Mendoza, María Mercedes
A1 Ortego, Felix
A1 García de Lacoba, María
A1 Magaña, Cristina
A1 Poza, Marta de la
A1 Farinós, Gema
A1 Castañera, Pedro
A1 Hernández-Crespo, Pedro
AB 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 optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.
PB Elsevier
SN 0965-1748
YR 2005
FD 2005
LK https://hdl.handle.net/20.500.14352/95233
UL https://hdl.handle.net/20.500.14352/95233
LA eng
NO Díaz-Mendoza, M., et al. «Diversity of Trypsins in the Mediterranean Corn Borer Sesamia Nonagrioides (Lepidoptera: Noctuidae), Revealed by Nucleic Acid Sequences and Enzyme Purification». Insect Biochemistry and Molecular Biology, vol. 35, n.o 9, septiembre de 2005, pp. 1005-20. https://doi.org/10.1016/j.ibmb.2005.04.003.
NO AcknowledgmentsWe 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).
NO European Commission
NO Ministerio de Educación y Ciencia (España)
DS Docta Complutense
RD 29 abr 2025