Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods

doi:10.1016/S0167-4838(00)00165-5

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1482, Issues 1–2, 18 October 2000, Pages 110-118

Biochimica et Biophy...

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Abstract

Recent gene sequence and crystal structure determinations of salivary proteins from several blood-sucking arthropods have revealed an unusual evolutionary relationship: many such proteins derive their functions from lipocalin protein folds. Many blood-sucking arthropods have independently evolved the ability to overcome a host organism’s means of preventing blood loss (called hemostasis). Most blood feeders have proteins that induce vasodilation, inhibit blood coagulation, and reduce inflammation, but do so by distinctly different mechanisms. Despite this diversity, in many cases the antihemostatic activities in such organisms reside in proteins with lipocalin folds. Thirteen such lipocalins are described in this review, with a particular focus on the heme-containing nitrophorins from Rhodnius prolixus, which transport nitric oxide, sequester histamine, and disrupt blood coagulation. Also described are the antiplatelet compounds RPAI, moubatin, and pallidipin from R. prolixus, Ornithodoros moubata, and Triatoma pallidipennis; the antithrombin protein triabin from T. pallidipennis; and the tick histamine binding proteins from Rhipicephalus appendiculatus.

Section snippets

Blood feeding and the spread of Chagas’ disease

Rhodnius prolixus, a small insect prevalent in South America but also found in North America, feeds on the blood of rodents and larger mammals, including humans. The insect is a relatively slow feeder, and probes the tissue until a suitable internal wound is created, or a blood vessel is tapped [1]. Like most blood-sucking insects, Rhodnius secretes a protein-rich saliva to aid in the taking of a blood meal. Nine salivary proteins have been characterized from Rhodnius, and all nine have

Nitrophorin structure

The initial isolation and characterization of the nitrophorins was accomplished by the fractionation of hundreds of pairs of Rhodnius salivary glands obtained through painstaking insect dissections. All four nitrophorins were identified from this source, and all three antihemostatic activities (NO transport, histamine binding, and anticoagulation) were discovered [11], [12], [13], [14], [20], [21], [22]. The subsequent cloning and expression of all four nitrophorins led to more detailed

Nitrophorin delivery of nitric oxide

Nitric oxide has long been of interest to chemists for its use as a spectroscopic probe, for the wide range of coordination chemistry it allows, and for its usefulness in molecular orbital theory (recently reviewed in [34]). More recently, biologists have found NO to be a signaling molecule in virtually all vertebrate cells and possibly all eukaryotic cells. The choice by nature of NO as a signaling molecule is curious since it is extremely reactive, due to an unpaired, antibonding electron,

Histamine binding by salivary proteins from R. prolixus and tick

Slow feeding blood-sucking insects such as ticks and R. prolixus face a difficulty: the tissue damage they cause results in inflammation and their salivary proteins illicit an immune response, which can lead to the ‘walling off’ of the affected area, or to the detection and execution of the insect. To overcome this problem these insects sequester histamine, the signaling molecule secreted by the host’s mast cells in response to tissue trauma. R. appendiculatus [5] and R. prolixus [22], [27]

Anticoagulation by NP2 and triabin

While feeding, blood-sucking insects must prevent blood coagulation so that blood will flow from the wound and not clog the insect’s mouth parts and gut. The saliva of R. prolixus has long been known to act as an anticoagulant [13]. Interestingly, this activity resides in NP2, also called prolixin-S, and thus provides a third activity in the nitrophorin lipocalin fold [12], [14]. Like all of the Rhodnius antihemostatic activities, NP2 acts early in the blood coagulation cascade. Exposure of

Antiplatelet activity in R. prolixus, T. pallidipennis, and Ornithodoros moubata

At the site of an injury, platelets are activated and adhere to exposed collagen in the blood vessel wall [44]. Binding directly activates platelets by a receptor-mediated signal transduction pathway, inducing pronounced alterations in platelet shape and biochemistry. Activated platelets secrete factors such as thromboxane A₂ and ADP that promote further platelet aggregation, and provide the phospholipid surface necessary for the blood-coagulation cascade. The extent of platelet aggregation is

Evolution and pharmacology

Thus, a curious story is unfolding concerning the means by which insects have gained the ability to feed on blood. This ability has independently evolved in several species, and generally the same steps in hemostasis are targeted by proteins in the insects’ saliva [3]. The lipocalin fold appears to be a protein scaffold of choice for the development of antihemostatic functions in insects, based on the prevalence of this fold in multiple blood-feeding organisms, and 13 such proteins are known (

Acknowledgements

We thank Dr. Guido Paesen for sending us the HBP2 coordinates prior to release from the protein data bank. This work was supported in part by NIH grants HL62969 and GM58727, and ADCRC grant 1-208A.

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Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-β-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-β-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O₂/NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-β-barrel heme-proteins.
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The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm⁻¹, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.
Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin
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Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (K_d = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.
Nitrosylation of ferric zebrafish nitrobindin: A spectroscopic, kinetic, and thermodynamic study
2022, Journal of Inorganic Biochemistry
Citation Excerpt :
Nitrophorins, which are present only in the salivary glands of blood-feeding insects Rhodnius prolixus and Cimex lectularius, release NO to the feeding site inducing vasodilation and counteracting the host hemostatic response. After NO release, nitrophorins bind histamine with high affinity displaying antihistaminic activity and impairing itch [25–31]. Although the function(s) of Nbs is still obscure, it has been postulated that they participate to NO signaling and metabolism; moreover, they catalyze peroxynitrite scavenging [11,13,31–33].
Nitrobindins (Nbs) are all-β-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO₂, and isomerizing peroxynitrite to NO₃⁻ and NO₂⁻. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV–Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH ≤ 7.0, Dr-Nb(III) binds reversibly NO, whereas at pH ≥ 7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (∼ 1.5 ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., k_lim) increase linearly with the OH⁻ concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH⁻-based catalysis. From the dependence of k_lim on [OH⁻], the value of the second-order rate constant k_OH− was obtained (5.2 × 10³ M⁻¹ s⁻¹). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO_2, and the other being tightly bound to the heme-Fe(II) atom.
Dissociation of the proximal His-Fe bond upon NO binding to ferrous zebrafish nitrobindin
2022, Journal of Inorganic Biochemistry
Nitrobindins (Nbs) are all-β-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O₂ metabolism, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pH 5.5 and 9.1 at 22.0 °C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6 × 10⁶ M⁻¹ s⁻¹ and 2.3 × 10⁶ M⁻¹ s⁻¹ and between 5.3 × 10⁻² s⁻¹ and 8.2 × 10⁻² s⁻¹, respectively. Interestingly, both UV–Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.
Oxygen-mediated oxidation of ferrous nitrosylated nitrobindins
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The O₂-mediated oxidation of all-β-barrel ferrous nitrosylated nitrobindin from Arabidopsis thaliana (At-Nb(II)-NO), Mycobacterium tuberculosis (Mt-Nb(II)-NO), and Homo sapiens (Hs-Nb(II)-NO) to ferric derivative (At-Nb(III), Mt-Nb(III), and Hs-Nb(III), respectively) has been investigated at pH 7.0 and 20.0 °C. Unlike ferrous nitrosylated horse myoglobin, human serum heme-albumin and human hemoglobin, the process in Nb(II)-NO is mono-exponential and linearly dependent on the O₂ concentration, displaying a bimolecular behavior, characterized by k_on = (6.3 ± 0.8) × 10³ M⁻¹ s⁻¹, (1.4 ± 0.2) × 10³ M⁻¹ s⁻¹, and (3.9 ± 0.5) × 10³ M⁻¹ s⁻¹ for At-Nb(II)-NO, Mt-Nb(II)-NO, and Hs-Nb(II)-NO, respectively. No intermediate is detected, indicating that the O₂ reaction with Nb(II)-NO is the rate-limiting step and that the subsequent conversion of the heme-Fe(III)-N(O)OO⁻ species (i.e., N-bound peroxynitrite to heme-Fe(III)) to heme-Fe(III) and NO₃⁻ is much faster. A similar mechanism can be invoked for ferrous nitrosylated human neuroglobin and rabbit hemopexin, in which the heme-Fe(III)-N(O)OO⁻ species is formed as well, although the rate-limiting step seems represented by the reshaping of the six-coordinated heme-Fe(III) complex. Although At-Nb(II)-NO and Mt-Nb(II)-NO are partially (while Hs-Nb(II)-NO is almost completely) penta-coordinated, density functional theory (DFT) calculations rule out that the cleavage of the proximal heme-Fe-His bond in Nb(II)-NO is responsible for the more stable heme-Fe(III)-N(O)OO⁻ species. Moreover, the oxidation of the penta-coordinated heme-Fe(II)-NO adduct does not depend on O₂ binding at the proximal side of the metal center. These features may instead reflect the peculiarity of Nb folding and of the heme environment, with a reduced steric constraint for the formation of the heme-Fe(III)-N(O)OO⁻ complex.

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ReviewNitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods

Abstract

Section snippets

Blood feeding and the spread of Chagas’ disease

Nitrophorin structure

Nitrophorin delivery of nitric oxide

Histamine binding by salivary proteins from R. prolixus and tick

Anticoagulation by NP2 and triabin

Antiplatelet activity in R. prolixus, T. pallidipennis, and Ornithodoros moubata

Evolution and pharmacology

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

Insect Biochem. Mol. Biol.

Structure

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Am. Heart J.

Trends Pharmacol. Sci.

Blood Rev.

Studies on the methods of feeding of blood sucking arthropods. I. The manner in which triatomine bugs obtain their blood meal, as observed in the tissues of the living rodent, with some remarks on the effects of the bite on human volunteers

Ann. Trop. Med. Parasitol.

Biochemical insights derived from diversity in insects

Annu. Rev. Biochem.

Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug.

Proc. Natl. Acad. Sci. USA

NITRIC OXIDE: a physiologic messenger molecule

Annu. Rev. Biochem.

The human inflammatory response

J. Cardiovasc. Pharmacol.

Platelets and inflammation: role of platelet-derived growth factor, adhesion molecules and histamine

Inflamm. Res.

Nitrophorin-2: a novel mixed-type reversible specific inhibitor of the intrinsic factor-X activating complex

Biochemistry

Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus

Thromb. Haemost.

Prolixin-S and prolixin-G: two anticoagulants from Rhodnius prolixus STAHL

Nature

Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus

Biochem. J.

Salivary apyrase of Rhodnius prolixus. Kinetics and purification

Biochem. J.

Review
Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods