Rhodnius

The Nitric Oxide-Releasing Heme Proteins from the Saliva of the Blood-Sucking Insect Rhodnius prolixus F. Ann Walker and William R. Montfort... 

Rhodniin 11 too Rhodnius prolixus Binds to and inhibits thrombin... 

This family contains species in the genera Triatoma, Rhodnius, and Panstron-gylus that are of major importance as vectors of Chagas disease, a debilitat-... 

THE NITRIC OXIDE-RELEASING HEME PROTEINS FROM THE SALIVA OF THE BLOOD-SUCKING INSECT Rhodnius prolixus... 

C. Early Work on the Nitrophorins from Rhodnius prolixus... 

NO-RELEASING HEME PROTEINS FROM Rhodnius prolixus SALIVA... 

Fig. 1. Nitric oxide (NO) synthesis by nitric oxide synthase (NOS) (upper left), NO reaction with soluble guanylate cyclase (sGC) (middle), and formation of cyclic GMP, which causes tissue-specific signaling (right). The roles of the salivary nitrophorins from Rhodnius prolixus in storing and releasing NO and binding histamine are included (lower left).

Fig. 2. Simplified illustration of the steps in blood coagulation inhibited by the salivary proteins of Rhodnius prolixus. The inhibited steps are indicated with crossed arrows. SAPLl-3 are also called RPAIl-3.

Fig. 3. EPR spectra of 100 pairs of Rhodnius salivary glands homogenized in 125 xl of phosphate-buffered saline at pH 7.2 (A) before argon equilibration (B) after equilibration in an argon atmosphere for 4 h (C) after equilibration of (B) with NO for 2 min. (D) difference spectrum, that is B — C. (E) homogenate as in (B) treated with dithionite (DT) to reduce Fe(III) to Fe(II), followed by equilibration with NO for 2 min. (The small signal at g = 2 in A-C is due to copper oxide in the liquid helium which had been condensed at the University of Arizona in a copper-plumbed helium liquiflcation apparatus ) All spectra are plotted on the same scale except (E), which is reduced in amplitude by a factor of 3. Reproduced with permission from Ref 24).

Fig. 4. Protein sequences of Rhodnius NPl-4 obtained from the gene sequences (45) and confirmed for NPl, NP2, and NP4 by X-ray ciystallography. Identical residues are marked. Alpha and beta structural features are also marked, as is the A-B loop that becomes ordered upon binding of NO (see Section III).

The initial procedures involved in expression of the Rhodnius nitrophorins in Escherichia coli were worked out for NPl by Drs. Donald E. Champagne and John F. Andersen at the University of Arizona during the spring and summer of 1996, and the purification protocols were... 

Fig. 5. UV-visible spectra of recombinant Rhodnius NP4 (pH 8.0) (a) without ligand (solid line) and NO complex (dashed line) (b) without ligand (solid line) and histamine complex (dashed line). Reproduced with permission from Ret (48).

Fig. 6. UV-visible spectra of 0.05 mM oxidized and reduced recombinant Rhodnius NP3 (a) at pH 7.5 without ligand (b) at pH 7.5 bound to NO (c) at pH 5.5 bound to NO. In each case, the spectrum of the oxidized nitrophorin is represented by a solid line and the reduced by a dashed line. Spectra were recorded in an optically transparent thin-layer electrochemical cell of approximate window thickness 0.05 mm. To obtain the fully oxidized and reduced spectra, potentials (vs Ag/AgCl) were applied until no change in optical spectrum occurred, of -1-600 and —400 mV, respectively (a), -1-200 and —400 mV, respectively (b), and 0 and -280mV, respectively (c).

Fig. 9. The continuous wave EPR spectrum of recombinant Rhodnius prolixus NPl-histamine at a microwave frequency of 9.338 GHz (trace 1) and field-sweep ESE spectra at 8.706 GHz (trace 2), 3.744 GHz (trace 3), 3.065 GHz (trace 4). Dashed arrows show the changes in magnetic fields corresponding to principalg -values at the different microwave frequencies. Inset trace 1, the primary ESE decay recorded at 8.706 GHz, Bo = 213 mT (the low-field turning point in the field-sweep ESE spectrum shown by trace 2 in the main panel). Inset trace 2, the primary ESE decay recorded at 3.065 GHz, Bo = 140.8 mT (the high-field turning point in the field-sweep ESE spectrum shown by trace 4 in the main panel). Reproduced with permission from Ref. (89).

The initial crystals of NPl were obtained with protein painstakingly isolated from Rhodnius salivary glands by the Ribeiro laboratory (44). This group also cloned all four nitrophorins (44, 45), which unfortunately resulted only in inclusion bodies or degraded protein in various... 

General Information about Rhodnius Nitrophorin Structures... 

We have completed several structures each of NPl, NP2, and NP4 (31, 46 9, 110). These structures reveal the Rhodnius nitrophorins to have a fold dominated by an eight-stranded antiparallel beta-barrel, as shown in Fig. 15, and to rely on a remarkable ligand-induced conformational change for NO transport, described later. The structures confirm that the nitrophorins are completely unrelated to the globins, the only other heme-based gas transport proteins whose structures are known. Rather, their fold places them in the lipocalin family, for which several other examples are known (111-113). Our initial nitrophorin structure was of NPl and was determined using standard MIR and... 

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