D7r4 is a short-form salivary protein from female Anopheles gambiae mosquitoes that facilitates blood feeding by binding and sequestering host biogenic amines (serotonin, histamine, norepinephrine, and tryptamine). It functions as a "kratagonist" - capturing host signaling molecules with high affinity to prevent their vasoconstrictive and inflammatory effects. The crystal structure of D7r4 has been solved (PDB: 2PQL, 2QEB, 2QEH, 2QEO, 2QEV) at high resolution (2.0-2.31 Angstroms), revealing a modified OBP-like fold with eight alpha-helices, three disulfide bonds, and a binding pocket adapted to amine ligands. D7r4 is secreted into saliva during blood feeding and belongs to the PBP/GOBP (Pheromone/Odorant-Binding Protein) structural family but has evolved a distinct function unrelated to olfaction.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005615 extracellular space | IBA GO_REF:0000033 | ACCEPT | Summary: This annotation correctly describes D7r4's localization based on phylogenetic inference. D7r4 is a secreted salivary protein with a signal peptide (residues 1-21) that is released into the extracellular space during blood feeding [UniProt Q7PNF2]. Reason: D7r4 is confirmed to be a secreted protein localized to female saliva. UniProt states "SUBCELLULAR LOCATION: Secreted" with evidence from PMID:17913537. The IBA annotation from phylogenetic trees is consistent with the IDA evidence for the same term. The protein contains a signal peptide and is found in the extracellular salivary compartment from which it is injected into the host during blood feeding [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 -!- SUBCELLULAR LOCATION: Secreted PMID:17913537 A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals |
| GO:0007608 sensory perception of smell | IBA GO_REF:0000033 | REMOVE | Summary: This annotation is INCORRECT. D7r4 is expressed exclusively in female salivary glands and functions in blood feeding, not in olfactory perception. The IBA annotation was inferred from homology to Drosophila odorant-binding proteins, but D7 proteins have evolved a completely different function in mosquito saliva. Reason: D7r4 is expressed specifically in female salivary glands, not olfactory tissues [PMID:12062411, PMID:11841502]. UniProt explicitly states the tissue specificity. The D7 proteins have evolutionarily repurposed the OBP-like fold from olfactory function to biogenic amine binding in saliva [PMID:16301315]. The IBA annotation appears to be based on structural family membership (PBP/GOBP) shared with Drosophila odorant-binding proteins, but this does not predict function. D7r4 binds serotonin, histamine, and norepinephrine - not odorants [PMID:17928288]. Deep research confirms short-form D7s "predominantly bind biogenic amines" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 -!- TISSUE SPECIFICITY: Female saliva (at protein level) (PubMed:17913537). Female salivary gland (PubMed:12062411). Not detected in female carcass without salivary glands (PubMed:12062411). Not detected in male tissues PMID:16301315 The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein superfamily |
| GO:0005549 odorant binding | IEA GO_REF:0000002 | MODIFY | Summary: This annotation is based on structural family membership (PBP/GOBP family via InterPro IPR006170) but does not reflect the actual function of D7r4. The protein binds biogenic amines (serotonin, histamine, norepinephrine, tryptamine), not odorants, and has no role in olfaction. Crystal structures with bound ligands demonstrate this directly. Reason: D7r4 belongs to the PBP/GOBP structural family (InterPro IPR006170), which led to this IEA annotation. However, the D7 proteins have evolutionarily repurposed this fold for binding biogenic amines in saliva, not odorants in antennae. D7r4 is expressed exclusively in female salivary glands [PMID:12062411], not olfactory tissues. The actual molecular functions are serotonin binding (GO:0051378), histamine binding (GO:0051381), norepinephrine binding (GO:0051380), and tryptamine binding. Crystal structures of D7r4 with bound serotonin (PDB:2QEH), histamine (PDB:2QEB), norepinephrine (PDB:2QEO), and tryptamine (PDB:2PQL) demonstrate the actual binding specificity [PMID:17928288]. UniProt documents specific binding residues for each ligand. The GO:0005549 definition "Binding to an odorant, any substance capable of stimulating the sense of smell" does not apply - serotonin, histamine, norepinephrine, and tryptamine are not odorants. Deep research confirms short-form D7s "predominantly bind biogenic amines (serotonin/5-HT, histamine, epinephrine/norepinephrine)" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 Binds serotonin, noradrenaline, histamine and tryptamine UniProt:Q7PNF2 The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: This annotation correctly reflects the subcellular localization of D7r4 as a secreted salivary protein. The IEA annotation from UniProt subcellular location vocabulary is appropriate. Reason: D7r4 is a secreted protein with a signal peptide (residues 1-21) [UniProt Q7PNF2]. It is released into the extracellular space (saliva) during blood feeding. The UniProt entry confirms "SUBCELLULAR LOCATION: Secreted" with experimental evidence from PMID:17913537. This is an appropriate CC annotation at the broader level. Deep research confirms D7 proteins are "secreted into saliva at the skin interface during probing/feeding" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 -!- SUBCELLULAR LOCATION: Secreted |
| GO:0042311 vasodilation | IEA GO_REF:0000043 | ACCEPT | Summary: This annotation is technically correct but represents the downstream physiological effect rather than the direct biological process. D7r4 exhibits vasodilating activity by sequestering vasoconstrictors (serotonin, norepinephrine) from the host. Reason: UniProt confirms D7r4 "Exhibits vasodilating activity" [UniProt Q7PNF2]. The mechanism is indirect - D7r4 sequesters serotonin and norepinephrine, which are vasoconstrictors, thereby preventing vasoconstriction and allowing vasodilation at the bite site. PMID:16301315 states D7 proteins "bind serotonin with high affinity, as well as histamine and norepinephrine" and that "Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties." While the annotation captures a real physiological effect, it is a downstream consequence of the kratagonist mechanism. However, since this is documented in the literature, it should be retained. Deep research confirms biogenic amine scavenging "counteracts platelet aggregation, vasoconstriction, and nociception during feeding" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 Exhibits vasodilating activity (PubMed:16301315) PMID:16301315 Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties |
| GO:0097746 blood vessel diameter maintenance | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: This annotation is a parent term of vasodilation and is somewhat redundant. D7r4 affects blood vessel diameter by counteracting vasoconstriction through sequestration of host biogenic amines. Reason: This annotation is inferred from the UniProt keyword "Vasodilator" via GO_REF:0000043. While technically accurate as a broader term encompassing vasodilation, it is less informative than GO:0042311 (vasodilation) which is also annotated. The primary mechanism of D7r4 is sequestration of vasoconstrictive amines, with blood vessel diameter maintenance being a downstream physiological consequence. This should be kept as a non-core annotation since it accurately reflects a real biological effect but is not the primary function of the protein. Supporting Evidence: UniProt:Q7PNF2 Exhibits vasodilating activity (PubMed:16301315) |
| GO:0005615 extracellular space | IDA PMID:17913537 Antibody response against saliva antigens of Anopheles gambi... | ACCEPT | Summary: This is high-quality experimental evidence from mass spectrometry identification of D7r4 in female mosquito saliva. This annotation should be accepted as representing the experimentally determined localization. Reason: PMID:17913537 provides direct experimental evidence (IDA - Inferred from Direct Assay) for D7r4 localization in the extracellular space (saliva). The study performed proteomic analysis of Anopheles gambiae saliva and identified D7r4 by mass spectrometry. UniProt confirms "Female saliva (at protein level) (PubMed:17913537)" [UniProt Q7PNF2]. This experimental evidence strongly supports the CC annotation. Supporting Evidence: PMID:17913537 A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals UniProt:Q7PNF2 -!- TISSUE SPECIFICITY: Female saliva (at protein level) (PubMed:17913537) |
| GO:0051378 serotonin binding | IDA PMID:17928288 The crystal structure of D7r4, a salivary biogenic amine-bin... | NEW | Summary: D7r4 binds serotonin with high affinity as demonstrated by direct binding assays and crystal structure with bound serotonin (PDB:2QEH). This is a core molecular function. Reason: UniProt confirms "Binds serotonin, noradrenaline, histamine and tryptamine (PubMed:16301315, PubMed:17928288)" [UniProt Q7PNF2]. The crystal structure of D7r4 with bound serotonin (PDB:2QEH) at 2.10 Angstrom resolution demonstrates the binding mode directly [PMID:17928288]. Specific binding residues are documented: Glu-28, His-56, Tyr-115, Asp-132, and Glu-135. Mutagenesis studies confirm these residues are critical for serotonin binding - E28L and E135L mutations significantly reduce serotonin binding [UniProt Q7PNF2]. GO:0051378 definition "Binding to serotonin (5-hydroxytryptamine), a monoamine neurotransmitter" accurately describes this function. PMID:16301315 states "four of these five short D7 proteins and the D7 long form bind serotonin with high affinity". Supporting Evidence: UniProt:Q7PNF2 Binds serotonin, noradrenaline, histamine and tryptamine (PubMed:16301315, PubMed:17928288) UniProt:Q7PNF2 The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae PMID:16301315 four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine |
| GO:0051381 histamine binding | IDA PMID:17928288 The crystal structure of D7r4, a salivary biogenic amine-bin... | NEW | Summary: D7r4 binds histamine with high affinity as demonstrated by direct binding assays and crystal structure with bound histamine (PDB:2QEB). This is a core molecular function. Reason: UniProt confirms "Binds serotonin, noradrenaline, histamine and tryptamine (PubMed:16301315, PubMed:17928288)" [UniProt Q7PNF2]. The crystal structure of D7r4 with bound histamine (PDB:2QEB) at 2.00 Angstrom resolution demonstrates the binding mode directly [PMID:17928288]. Specific binding residues are documented: Tyr-115, Asp-132, and Glu-135. UniProt states D7r4 "Inhibits histamine-, serotonin- and partially noradrenaline-induced smooth muscle contraction" [UniProt Q7PNF2]. GO:0051381 definition "Binding to histamine, a physiologically active amine" accurately describes this function. PMID:16301315 demonstrates histamine binding activity. Supporting Evidence: UniProt:Q7PNF2 Inhibits histamine-, serotonin- and partially noradrenaline-induced smooth muscle contraction (PubMed:16301315) UniProt:Q7PNF2 The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae |
| GO:0051380 norepinephrine binding | IDA PMID:17928288 The crystal structure of D7r4, a salivary biogenic amine-bin... | NEW | Summary: D7r4 binds norepinephrine as demonstrated by direct binding assays and crystal structure with bound norepinephrine (PDB:2QEO). This is a core molecular function. Reason: UniProt confirms "Binds serotonin, noradrenaline, histamine and tryptamine (PubMed:16301315, PubMed:17928288)" [UniProt Q7PNF2]. The crystal structure of D7r4 with bound norepinephrine (PDB:2QEO) at 2.31 Angstrom resolution demonstrates the binding mode directly [PMID:17928288]. Specific binding residues are documented: Glu-28, Phe-43, Asp-132, and Glu-135. UniProt states D7r4 "Inhibits histamine-, serotonin- and partially noradrenaline-induced smooth muscle contraction" [UniProt Q7PNF2]. GO:0051380 definition "Binding to norepinephrine" accurately describes this function. PMID:16301315 states D7 short proteins bind "norepinephrine" with high affinity. Supporting Evidence: UniProt:Q7PNF2 Binds serotonin, noradrenaline, histamine and tryptamine (PubMed:16301315, PubMed:17928288) UniProt:Q7PNF2 The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae |
| GO:0035821 modulation of process of another organism | IDA PMID:16301315 Function and evolution of a mosquito salivary protein family | NEW | Summary: D7r4 modulates host hemostatic and inflammatory responses during blood feeding by sequestering host biogenic amines. This is an appropriate BP annotation for the cross-species effect. Reason: D7r4 clearly modulates processes in the vertebrate host. UniProt states "Modulates blood feeding of female mosquitoes on vertebrate species by binding and sequestering different mediators involved in the host response" [UniProt Q7PNF2]. The GO:0035821 definition "A process in which an organism effects a change in a biological process in another organism" accurately captures this function. By sequestering serotonin, histamine, and norepinephrine from the host, D7r4 prevents vasoconstriction, platelet aggregation, and inflammation. PMID:16301315 confirms "Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties." Deep research describes D7 proteins as "kratagonists - high-affinity scavengers of host small-molecule inflammatory and hemostatic mediators" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: UniProt:Q7PNF2 Modulates blood feeding of female mosquitoes on vertebrate species by binding and sequestering different mediators involved in the host response PMID:16301315 Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties |
| GO:1900047 negative regulation of hemostasis | IDA PMID:16301315 Function and evolution of a mosquito salivary protein family | NEW | Summary: D7r4 negatively regulates host hemostasis by sequestering biogenic amines that promote platelet aggregation and vasoconstriction. This is a core biological process annotation. Reason: By sequestering serotonin (which promotes platelet aggregation) and norepinephrine (a vasoconstrictor), D7r4 negatively regulates the host hemostatic response at the bite site. PMID:16301315 states "Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties." GO:1900047 definition "Any process that stops, prevents or reduces the frequency, rate or extent of hemostasis" accurately captures this function. Deep research confirms biogenic amine scavenging "counteracts platelet aggregation, vasoconstriction, and nociception during feeding" [file:ANOGA/D7r1/D7r1-deep-research-falcon.md]. Supporting Evidence: PMID:16301315 Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties |
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Download this section (compressed HTML)Q: What are the binding affinities (Kd values) of D7r4 for each of its ligands (serotonin, histamine, norepinephrine, tryptamine), and does it show preference for one over others?
Suggested experts: Andersen JF, Mans BJ, Ribeiro JM
Q: Do the different D7 short forms (D7r1-5) have distinct ligand binding specificities that enable non-redundant function during blood feeding?
Suggested experts: Calvo E, Mans BJ
Q: What is the structural basis for D7r4's ability to bind multiple chemically distinct biogenic amines in the same binding pocket?
Suggested experts: Andersen JF, Mans BJ
Experiment: Generate D7r4 knockout A. gambiae using CRISPR and measure blood meal size, feeding time, feeding success rate, and host inflammatory responses compared to wild-type controls
Hypothesis: D7r4 knockout mosquitoes will have reduced blood feeding efficiency due to enhanced host hemostatic and inflammatory responses
Type: Gene knockout and feeding assay
Experiment: Perform comparative structural analysis and ancestral sequence reconstruction to determine which amino acid changes are responsible for the shift from odorant to biogenic amine binding
Hypothesis: D7r4 has evolved from odorant-binding proteins through changes in ligand binding specificity
Type: Structural biology and molecular evolution
Experiment: Perform competitive binding assays with all D7 short forms (D7r1-5) against a panel of biogenic amines to determine the binding specificity landscape of the D7 short form family
Hypothesis: D7r4 and other D7 short forms have non-overlapping ligand preferences that together provide comprehensive sequestration of host mediators
Type: Binding assays and functional complementation
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