Purification and cloning of the salivary nitrophorin from the hemipteran Cimex lectularius.

Cimex lectularius and Rhodnius prolixus contain salivary nitric oxide (NO) that may help them to feed on their vertebrate hosts by promoting vasodilation and inhibiting platelet aggregation. Salivary NO is associated with heme proteins (nitrophorins) that store and transport NO from the insect salivary glands to the skin of the host. In this study, the salivary nitrophorin of Cimex lectularius was purified by DEAE chromatography and reverse-phase high-performance liquid chromatography. The purified nitrophorin had a molecular mass of 32.9 kDa. The DEAE-purified hemoprotein was able to bind NO, and this binding shifted the absorption maximum from 388 nm to 438 nm. The ratio of heme to apoprotein was estimated to be of 1:1. A cDNA clone of 1079 base pairs was sequenced and was found to code for a protein with a molecular mass of 31.7 kDa. The clone sequence was in agreement with the internal peptide sequences obtained from the purified protein. Sequencing of the isolated clone indicates high similarity to several inositol phosphatases; however, no significant similarities emerged when the sequence of C. lectularius nitrophorin was compared with that of R. prolixus nitrophorin, the only other nitrophorin known in insect saliva. Because C. lectularius and R. prolixus belong to two different families of Hemiptera that evolved independently to blood feeding, a case is made for the convergent evolution of these two insect nitrophorins.

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