ABSTRACT
A cDNA encoding a trypsin-like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin-like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.
Subject(s)
Feeding Behavior , Panstrongylus/enzymology , Protein Processing, Post-Translational , Salivary Glands/enzymology , Serine Proteases/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Feeding Behavior/drug effects , Fibrinolysis/drug effects , Gene Expression Profiling , Gene Expression Regulation/drug effects , Hydrolysis/drug effects , Larva/drug effects , Larva/enzymology , Molecular Sequence Data , Molecular Weight , Panstrongylus/drug effects , Panstrongylus/genetics , Protein Processing, Post-Translational/drug effects , Salivary Glands/drug effects , Sequence Alignment , Serine Proteases/chemistry , Serine Proteases/isolation & purification , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Substrate Specificity/drug effects , Time FactorsABSTRACT
Antibacterial proteins like lysozyme are important components of the insect non-specific immune response against bacteria. The complementary deoxyribonucleic acid (cDNA) encoding a new lysozyme from Triatoma infestans, named lysozyme2, has been amplified by polymerase chain reaction and the rapid amplification of cDNA ends technique. The gene is expressed in the small intestine of the insect. The deduced protein sequence shows up to 70% similarity to lysozymes from other species. Furthermore, the protein exhibits significant structural concordance to other insect lysozymes. A striking feature of the lysozyme2 protein is the replacement of the conserved amino acid residues of the active site of classical c-type lysozymes, glutamate and aspartate, by valine and tyrosine.
