cDNA Cloning, Expression and Characterization of Nitric‐oxide Synthase from the Salivary Glands of the Blood‐Sucking Insect Rhodnius prolixus

Abstract

Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH,), calmodulin, FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.Keywords : insect; salivary gland ; nitric-oxide synthase; CaZ+ and calmodulin dependence; baculovirus.NO is produced in various mammalian tissues where it plays physiologically important roles [I]. NO acts as neurotransmitter in the peripheral and the central nervous systems and is involved in brain development, memory formation and learning. In the circulatory system, NO is produced in endothelial cells, and acts as a regulator of vascular tone and platelet aggregation. NO is induced in macrophages, neutrophils and liver cells, and acts as an important effector of the cell-mediated immune system. The enzyme critical for NO production, NO synthase (NOS), is found in these different tissues, and various isofoms have been purified and characterized, and some have been cloned [2, 31. Mammalian NOS have been grouped into two general categories: constitutive (Ca*' kalmodulin dependent, e.g. endothelial NOS and neuronal NOS) and inducible (Ca"/calmodulin independent, e.g. macrophage) types.By contrast, in invertebrates, the production and utilization of NO is not as well understood, although similar physiological functions are probably present. In some insects and molluscs, the existence of NOS in brain has been demonstrated, and the function of NO as a neurotransmitter was suggested [4, 51. In the silkworm, Bombyx mori, NOS activity in the fat body was induced by lipopolysaccharide, an agent that mimics bacterial assault, which suggested the involvement of NO in the insect immune defense system [ 3 ] . This activity required Ca2+, calmodulin, NADPH, FAD, FMN and tetrahydrobiopterin (BH,), similarly to mammalian NOS. Correspondence to M . Yuda, Department of Medical Zoology,Fux: f 8 l 592 31 5215. Abhreviufions. NO, nitric oxide; NOS, nitric-oxide synthase; RACE, rapid amplification of cDNA ends ; BH,, tetrahydrobiopterin ; MOI, multiplicity of infection.Note. The nucleotide sequence data published here have been submitted to the GenBank database and ar...

Distribution and characterization of nitric oxide synthase in the nervous system of Triatoma infestans (Insecta: Heteroptera)

“…Western blotting has revealed the presence of a stained band at 134 kDa in the nervous tissue of the CNS in T. infestans; this is in good agreement with the molecular weight of 132 kDa reported for NOS of the salivary gland of R. prolixus (Yuda et al 1996). These results are also in agreement with previous reports in non-triatomine species (Elphick et al 1995;Nighorn et al 1998).…”

“…These results are also in agreement with previous reports in non-triatomine species (Elphick et al 1995;Nighorn et al 1998). The presence of other bands of lower molecular weights between 90 and 51.7 kDa is also in line with previous observations in salivary gland homogenates (Yuda et al 1996).…”

“…Highly sensitive immunocytochemistry protocols (Settembrini and Villar 2004) might be useful to determine whether these diverse distributions of NOS are linked to different chemosensory modalities. The recent availability of antibodies generated against a peptide sequence conserved in all known insect NOS (Regulski and Tully 1995;Yuda et al 1996;Nighorn et al 1998;Luckhart and Rosenberg 1999;Imamura et al 2002) and the use of secondary reagents that enhance the detection of the antigen-antibody reaction provide a sensitive and specific tool for the tissue localization of this enzyme. The highly specific immunolocalization of the enzyme has been shown to match sites of in-situ hybridization for NOS mRNA but lacks the limitations inherent in the latter technique (Hsu et al 1981;Shu et al 1988;Gibson and Nighorn 2000).…”

Reductive genome evolution, host–symbiont co-speciation and uterine transmission of endosymbiotic bacteria in bat flies

“…The generic name honors Manfred Aschner, who first described the endosymbionts of nycteribiid bat flies (Aschner, 1931(Aschner, , 1946. The specific name is after a Japanese biologist Yasuo Chinzei, who significantly contributed to the biochemistry and molecular biology of blood-sucking insects (Yuda et al, 1996;Kadota et al, 2004).…”

Nitrophorins and nitrobindins: structure and function

“…Rp-NPs possess anti-hemostatic activity, storage, transport and release of NO and binds host histamine (26,27,37). Of note, Rp-NPs are synthesized in the salivary glands where NO is produced from a NO-synthase similar to mammalian constitutive isoforms (46). Although not evolutionarily related to Rp-NPs, the Cl-NP also transports, stores and releases NO by using a ferric heme-Fe.…”