Characterization and cDNA cloning of a hemoprotein in the salivary glands of the blood-sucking insect, Rhodnius prolixus.

Three major red hemoproteins, named RpSG I, II (identical with prolixin-S) and III, in the salivary glands of the blood-sucking insect, Rhonius prolixus, show homology in N-terminal amino acid (AA) sequences, and are immunologically related. We focussed on one of these proteins, RpSG-I, in this paper. RpSG-I in fresh salivary gland extract was separated into two components (Ia and Ib) by isoelectric focussing gel electrophoresis. Absorption spectra of RpSG-Ia and Ib showed Soret peaks at 400 nm and 420 nm, respectively, suggesting that they are nitric oxide (NO)-unbound and -bound hemoproteins and function as NO-carriers. RpSG-I is stage-specific in appearance, being absent in 3rd and 4th instar nymphs, appearing and increasing gradually in 5th (last) instar nymphs after engorgement, and present in the adult stage. We purified RpSG-I from salivary gland extract by size exclusion and ion exchange HPLCs. It is a single electrophoretic band with an absorption peak at 400 nm, representing the NO-unbound molecule. Full-size cDNA of RpSG-I was cloned by screening with a specific polyclonal antibody from a salivary gland cDNA library. Sequence analysis of RpSG-I cDNA showed an open reading frame encoding a signal peptide (23 AA) and mature protein (179 AA) of 19,778 daltons. The deduced N-terminal AA sequence of the RpSG-I was identical with that of the hemoprotein reported as nitrophorin-3 (Champagne et al., 1995).