CHARACTERIZATION AND CDNA CLONING OF A HEMOPROTEIN IN THE SALIVARY-GLANDS OF THE BLOODSUCKING INSECT, RHODNIUS-PROLIXUS

Three major red hemoproteins, named RpSG I. II (identical with prolixi n-S) and III, in the salivary glands of the blood-sucking insect, Rhod nius 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 sep arated into two components (Ia and Ib) by isoelectric focussing gel el ectrophoresis. Absorption spectra of RpSG-Ia and Ib showed Soret peaks at 400 nm and 330 nm, respectively, suggesting that they are nitric o xide (NO)-unbound and -bound hemoproteins and function as NO-carriers. RpSG-I is stage-specific in appearance, being absent in 3rd and 4th i nstar 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 sa livary gland cDNA library. Sequence analysis of RpSG-I cDNA showed an open reading frame encoding a signal peptide (23 AA) and mature protei n (179 AA) of 19778 daltons. The deduced N-terminal AA sequence of the RpSG-I was identical with that of the hemoprotein reported as nitroph orin-3 (Champagne et al., 1995). (C) 1998 Elsevier Science Ltd. All li ghts reserved.