An Overview Of D7 Protein Structure And Physiological Roles in Blood-Feeding Nematocera Part 1
Jun 14, 2023
Simple Summary:
Vectors are organisms that can transmit infectious pathogens from one host (human or animal) to another. Many vectors (including mosquitoes, sand flies, and ticks) have one common characteristic: they are blood-feeding (hematophagous) arthropods. Every time they bite their vertebrate host, skin and vascular injury triggers a series of responses that in place could lead to interruption of blood flow to their mouthparts, and to host awareness due to itching and pain. Nevertheless, their saliva contains a cocktail of molecules capable to counteract these host responses (hemostasis, inflammation, and immunity), allowing them to feed successfully.
Many times, the pathogens they transmit are injected into the host with saliva. Therefore, understanding the composition of vector saliva is crucial to study their biology, and their vectorial capacity, as well as to proposing new methods to control the diseases they transmit (including new vaccine candidates). Among important salivary protein families is the D7, abundantly expressed in bloodsucking Diptera and distantly related to Odorant-Binding Proteins (OBP). Here, we provide an extensive review of the D7 protein's structure, function, and evolution, discussing how gene duplication and modifications in their OBP-like domains lead to the gain and loss of function in different hematophagous Diptera species.
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Abstract:
Each time an insect bites a vertebrate host, skin and vascular injury caused by piercing triggers a series of responses including hemostasis, inflammation, and immunity. In place, this set of redundant and interconnected responses would ultimately cause blood coagulation, itching, and pain leading to host awareness, resulting in feeding interruption in the best-case scenario. Nevertheless, hematophagous arthropod saliva contains a complex cocktail of molecules that are crucial to the success of blood-feeding.
Among important protein families described so far in the saliva of blood-sucking arthropods, is the D7, abundantly expressed in blood-feeding Nematocera. D7 proteins are distantly related to insect Odorant-Binding Proteins (OBP), and despite low sequence identity, observation of structural similarity led to the suggestion that like OBPs, they should bind/sequester small hydrophobic compounds. Members belonging to this family are divided into short forms and long forms, containing one or two OBP-like domains, respectively. Here, we provide a review of the D7 protein's structure and function, discussing how gene duplication and some modifications in their OBP-like domains during evolution lead to the gain and loss of function among different hematophagous Diptera species.
Keywords:
D7 proteins; odorant-binding proteins; vector biology; hematophagy; vector saliva; hemostasis; inflammation.
1. Introduction
Vector-borne diseases are quite diverse in terms of symptoms, characteristics, etiologic agents, and their vectors. Despite this diversity, a common feature shared by most known vectors is the fact that they are hematophagous arthropods. The ability to feed on blood poses many challenges including the capacity to find the host, pierce its skin and find blood source, suck the blood to later digest it, and deal with oxidative stress generated by its digestion (reviewed in [1–3]).
Each time an insect bites its vertebrate host, skin and vascular injury triggers a series of responses including hemostasis, inflammation, and immunity [1,2,4–8]. These complex and redundant biological processes are interconnected and capable to propagate due to the production/secretion of many classes of mediators, including eicosanoids, biogenic amines, coagulation, and complement factors.
In place, they would cause an interruption of blood flow to the mouthparts of the hematophagous arthropod (due to vascular constriction, platelet aggregation, and blood coagulation) and trigger host awareness (due to itching and pain). Nevertheless, the saliva of blood-feeding arthropods contains an equally complex and redundant mix of molecules capable to counteract host defenses playing a pivotal role in the success of hematophagy (reviewed in [1,2,4–8]). Indeed, as stated before by Ribeiro, hematophagous arthropods are “live syringes” and their saliva is still a relatively unexploited source of pharmacologically active molecules [4].
The presence of anticoagulant molecules in hematophagous arthropods' saliva was first reported at the beginning of the last century [9,10], and the demonstration of the importance of salivary secretion in blood feeding was first proposed in the 800 s [11–13]. Yet, the composition of the saliva and the specific function of its components was still poorly understood. The use of large-scale sequencing and proteomics over the last decades allowed a better understanding of saliva composition but also revealed its complexity and how little is known [2,14]. Because hematophagy has arisen independently several times during arthropod evolution, many ways to deal with the challenges imposed by this habit appeared among the different insect orders, or even within the same genus leading to the wide variation in the repertoire of proteins to counteract host defenses to the bite [2,4,5,7,15,16].
Among important protein families described so far in the saliva of blood-sucking arthropods, is the D7, abundantly expressed in hematophagous Diptera [17–19] and distantly related to insect Odorant-Binding Proteins (OBP)/ Pheromone-Binding Proteins (PBP).
In insects, OBPs typically range from 100 to 160 residues, but as observed for some members belonging to “atypical/two domains” or “Plus C” sub-families described in Diptera, that can have up to 300 amino acids [20–22]. They are present in diverse orders and the number of annotated OBP genes varies a lot among different species, as well as the amino acid sequence.
Nevertheless, their cysteines are highly conserved—classically 6, although this number can vary from 4 in minus C OBPs to 8 in plus OBPs to even 9 or 10—positioned within the polypeptide chain with a signature distance between some of those [20,21,23]. Differently from vertebrate OBPs, which are all-β proteins belonging to the lipocalins superfamily (SCOP: 3001332), insect OBPs are classified as all-α proteins and belong to the insect pheromone/odorant-binding superfamily (SCOP ID: 4000957). Despite the low sequence identity between members, classically insect OBPs have a very characteristic tertiary structure, with 6 α-helices typically stabilized by 3 disulfide bonds forming a binding cavity (Figure 1A), surrounded by hydrophobic residues [20,24–26]. The great number and the diversity of their primary structure, while maintaining the overall architecture, made them very versatile in terms of ligands, but all with one characteristic in common: their ability/potential to bind small hydrophobic molecules.
D7 proteins, nonetheless, are restricted to blood-feeding Nematocera, where they are abundantly expressed in their salivary glands. Members of this family are classified in (1) short forms (D7S), also known as D7 related (D7r), with molecular weight around 15–17 kDa, containing one OBP-like domain (Figure 1B); (2) long forms (D7L), with molecular weights around 30–38 kDa, composed by two OBP like domains (Figure 1C). As first proposed by Arcà and colleagues [27] and later observed by X-ray diffraction crystallography [7,28–30], despite their low sequence similarities to OBPs, the D7 protein domains architecture is very similar to OBPs, being composed by α-helices (generally 7–8 α-helices though instead of 6), forming a hydrophobic pocket (Figure 1B, C) suitable to bind hydrophobic molecules, as will be further discussed below.
Most of the D7 proteins characterized so far were found in the saliva of blood-sucking Diptera, where they act as anti-hemostatic and/or anti-inflammatory molecules, facilitating blood feeding. In the present paper, we provide a comprehensive review of D7 and D7-like protein structure, function, and evolution, comparing some aspects with arthropod OBPs and other protein families relevant to vector physiology.
2. D7s: From the Description of the First Gene to Clues Regarding Their Diversity, Distribution, and Functions
The first gene encoding a D7 protein was isolated and described in 1991 in Aedes aegypti [31], shown to be abundantly and exclusively expressed in female salivary glands producing a 37 kDa protein. In situ hybridization using dissected female salivary glands probed with antisense RNA for the coding region of this gene revealed its expression was mainly in the distal lateral and medial lobes, regions that are very well differentiated in females (hematophagous) when compared to males (phytophagous), suggesting its product was very likely to be involved in blood feeding. Later, in a pioneering study aiming to identify genes that were expressed exclusively in the salivary gland of Anopheles gambiae and whose products contained signal peptides, six cDNAs were isolated.
Three of them, shown to be abundantly expressed in female salivary glands, encoded transcripts similar, albeit shorter, to the previously described Aedes aegypti D7, suggesting that this was a new protein family. All three aligned completely with the Aedes D7 C-terminal domain, therefore being named D7-related proteins (D7r): D7r1–D7r3 [32]. Likewise, they were expressed in female distal lateral lobes, although only one of them (D7r1) was expressed in medial lobes too [32].
A few years later, a fourth D7r (D7r4) was also found in Anopheles gambiae female salivary glands and shown to be located close to the other three forms forming a cluster on chromosome 3R [27]. When aligned, these 4 transcripts had similarities between them ranging from 53 to 73%, and very importantly, they had a low level of similarity to OBPs and Pheromone binding Proteins (PBP), but their 4 cysteines were located at conserved positions related to antennal and non-antennal OBPs. Despite low sequence similarity, secondary structure prediction suggested structural similarities to OBPs, hence it was postulated that D7s might also have a hydrophobic binding pocket surrounded by α-helices and be capable to bind or carry small hydrophobic molecules, probably mediators involved in host responses (like inflammation and hemostasis), given the fact that they were abundant and exclusive to female saliva [27].
The fact that these An. gambiae short forms aligned with the C-terminal of the Ae. aegypti D7L, and that Southern blot assays found other members in closely related species raised the possibility that members of this gene family might be present in other mosquito species and that proteins with different lengths encoded by these genes could have similar functions, but different targets due to difference in their primary structure [27].
Indeed, Suwan and coworkers reported for the first time genes encoding one D7L and 2 D7-related (short) forms in the salivary gland of Anopheles stephensi, and Western blot with polyclonal antibody produced to recognize the long form protein had cross reactivity also with D7rs [33]. This was the first report showing that indeed long and short forms could be found in the same species. Then, subsequent studies reported the presence of other members of this family in different mosquito (family Culicidae) species including Anopheles arabiensis, Aedes aegypti, and Anopheles darlingi [17,18], as well as in other hematophagous Diptera belonging to the family Psychodidae (sand flies) [17].
Altogether these studies strongly suggested that: (1) D7 was a family of proteins probably widespread in hematophagous Diptera, exclusively expressed in female salivary glands, therefore they might have an important role in blood feeding; (2) D7 proteins different in length and amino acid sequence were present in different species and within the same species, probably as a result of gene duplication generating diversity; (3) their putative targets should be small hydrophobic molecules, such as inflammation and/or hemostasis mediators [27,33]. Nevertheless, their function was still elusive.
3. Salivary D7 Proteins Are Anti-Inflammatory and Anti-Hemostatic Molecules
D7 and other OBP-like proteins present in the saliva of blood-sucking Nematocera can have variability in their primary structure, leading to gain and loss of function despite keeping some key features and general architecture, as has been observed in many OBPs. Table 1 summarizes D7 and D7-like proteins described so far in the saliva of different species, and their ligands and provides their Protein Data Bank (PDB) accession number, where applicable. Details and physiological relevance are discussed below.
3.1. Contact Pathway Inhibitors
The first D7 to have its function characterized was a short form (D7r), named Hamadarin [35], expressed in the SG of Anopheles stephensi females. Its closest D7 is expressed in An. gambiae would be D7r1. Hamadarin was shown to bind Factor XII (FXII) and High Molecular Weigh Kininogen (HMWK) therefore inhibiting contact pathway activation and consequently bradykinin production, acting as an anti-inflammatory molecule. Nevertheless, in both cases, the interaction with FXII and HMWK, studied by Surface Plasmon Resonance (SPR), occurred only in the presence of Zn2+ and did not affect activated factors amidolytic activity. Rather, its inhibitory effect on contact pathway activation was due to its interference with the reciprocal activation of FXII and kallikrein that should occur upon their interaction with charged surfaces [35]. A few years later another contact pathway activation inhibitor present in the saliva of Anopheles stephensi (Anophensin) was characterized and shown to have similar targets to Hamadarin but belonged to a completely different protein family [42].
Noteworthy, among the most expressed proteins in sandfly saliva [43–45], is a group of proteins that also belong to the OBP superfamily, are not found in mosquitoes, and are distinct from D7 proteins. They first got attention not only for their abundance but because they were identified as vaccine candidates against Leishmaniasis [46,47]. Only more than a decade later two members of this group were characterized in the saliva of Phlebotomus duboscqi: PdSP15a and PdSP15b (P. duboscqi Salivary Protein 15 a and b, respectively). Both are very similar to each other and inhibit contact pathway activation and bradykinin production [41] but through a mechanism distinct from that described for Hamadarin [35]. PdSP15a and b do not bind to any coagulation factor (zymogens or activated forms), kallikrein, or prekallikrein. Their action though is through binding negatively charged polymers such as dextran sulfate (DS), polyphosphate (PP), and heparin, preventing their interaction with FXII, necessary for its auto-cleavage that triggers its activation, and subsequent propagation of contact pathway activation through reciprocal activation of FXII and plasma prekallikrein (PK), as well as activation of FXI by thrombin and FXIIa. Importantly while classical OBPs and D7s have a hydrophobic pocket where their small hydrophobic ligand binds, structural data suggest its mechanism of action is through electrostatic interaction between its anionic surface and negatively charged ligands, rather than any binding inside a hydrophobic cavity [41,47].
The plasma coagulation cascade can be initiated by two distinct branches: (1) the extrinsic pathway, initiated when factor VII (FVII) is activated upon binding to subendothelial tissue factor (TF) exposed after vascular injury. (2) The contact pathway, also known as the intrinsic pathway, is initiated upon activation of FXII to FXIIa triggered by its contact with negatively charged surfaces. Both pathways after a series of reactions converge to the common pathway through FX activation that ultimately leads to fibrinogen cleavage to fibrin, essential for clot formation [48,49]. In recent years, drugs targeting intrinsic (contact) pathway components such as FXII and FXI have been extensively studied since it was shown that mice lacking FXII were protected from thrombus formation while having no major bleeding disorders [50–52], suggesting this pathway is important for pathological coagulation.
Upon activation, factor XII cleaves plasma prekallikrein (PK) generating kallikrein, that in addition to reciprocal activation with FXII also catalyzes the hydrolysis of HMWK generating bradykinin (kallikrein-kinin system) [53]. Bradykinin is a potent pro-inflammatory mediator that increases endothelial permeability [48,54] and pain [55]. Therefore, the presence of contact pathway inhibitors in the saliva of hematophagous arthropods would play an important role as anti-inflammatory molecules by reducing bradykinin production (as shown for the D7s/OBP-like proteins hamada in [35] and PdSP15s [41]), and by inhibiting/preventing plasma leakage induced by contact pathway activation as reported for PdSP15s [41].
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