D7L3 (AngaD7L3) is a long-form salivary D7 protein from Anopheles gambiae that uniquely binds serotonin among the A. gambiae D7 long-forms. This is unusual because long-form D7 proteins typically bind eicosanoids (leukotrienes, thromboxane A2), while biogenic amine binding is usually a short-form D7 function. D7L3 binds serotonin with high affinity but does not bind other biogenic amines (tryptamine, octopamine, dopamine, adrenaline) or eicosanoids (LTC4, LTD4, LTB4, thromboxane A2). By sequestering host serotonin at the bite site, D7L3 functions as a kratagonist that inhibits serotonin-induced platelet aggregation and vasoconstriction, thereby facilitating blood feeding.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0035821 modulation of process of another organism | IEA GO_REF:0000108 | ACCEPT | Summary: This annotation accurately describes D7L3's function. The protein modulates host hemostatic responses during blood feeding by sequestering serotonin, which prevents vasoconstriction and platelet aggregation. This is a correct BP annotation for the cross-species effect. Reason: D7L3 clearly modulates processes in the vertebrate host. The UniProt entry states it "Modulates blood feeding of female mosquitoes on vertebrate species by binding and sequestering different mediators involved in the host response" [UniProt A0A1S4HE90]. PMID:35460690 demonstrates that "AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction" - this is direct modulation of host hemostatic processes. The GO:0035821 definition "A process in which an organism effects a change in a biological process in another organism" accurately captures this kratagonist function. Supporting Evidence: UniProt:A0A1S4HE90 Modulates blood feeding of female mosquitoes on vertebrate species by binding and sequestering different mediators involved in the host response PMID:35460690 AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction |
| GO:0005549 odorant binding | IEA GO_REF:0000002 | REMOVE | Summary: This annotation is INCORRECT. D7L3 belongs to the PBP/GOBP structural family (InterPro IPR006170) which led to this IEA annotation, but D7 proteins have evolved distinct functions unrelated to olfaction. D7L3 binds serotonin, not odorants, and is expressed in salivary glands not olfactory tissues. Reason: D7L3 is a salivary protein that functions as a serotonin kratagonist, not an odorant-binding protein. The PBP/GOBP fold has been evolutionarily repurposed in D7 proteins for binding biogenic amines and eicosanoids in saliva. D7L3 is expressed in adult female salivary glands [UniProt A0A1S4HE90], not olfactory tissues. The actual molecular function is serotonin binding (GO:0051378). Serotonin (5-hydroxytryptamine) is a neurotransmitter/vasoactive amine, not an odorant. PMID:35460690 explicitly shows "AngaD7L3 binds serotonin" via isothermal titration calorimetry. This is a clear case where structural family membership does not predict function. Supporting Evidence: UniProt:A0A1S4HE90 Binds serotonin with high affinity PMID:35460690 AngaD7L3 binds (E) serotonin (5-HT). Titration curves are representative of at least three measurements. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: This annotation correctly reflects the subcellular localization of D7L3 as a secreted salivary protein. Reason: D7L3 is a secreted protein with a signal peptide (residues 1-26) [UniProt A0A1S4HE90]. It is released into the extracellular space (saliva) during blood feeding. The UniProt entry confirms "SUBCELLULAR LOCATION: Secreted" [UniProt A0A1S4HE90]. This is an appropriate CC annotation. Supporting Evidence: UniProt:A0A1S4HE90 SUBCELLULAR LOCATION: Secreted |
| GO:0042311 vasodilation | IEA GO_REF:0000043 | ACCEPT | Summary: This annotation requires clarification. D7L3 does exhibit vasodilating activity, but the mechanism is indirect - by sequestering serotonin, it prevents serotonin-induced vasoconstriction rather than directly causing vasodilation. This is still an accurate description of the physiological outcome. Reason: UniProt confirms "Exhibits vasodilating activity (PubMed:35460690)" [UniProt A0A1S4HE90]. PMID:35460690 demonstrates that AngaD7L3 is a vasodilator via myography assays, showing "A. gambiae salivary D7L1 and D7L3 proteins are vasodilators" with documented LogIC50 values. The mechanism is kratagonism - by sequestering serotonin, D7L3 prevents serotonin from causing vasoconstriction, resulting in relative vasodilation. The functional outcome is accurate even if the mechanism is indirect. Supporting Evidence: UniProt:A0A1S4HE90 Exhibits vasodilating activity PMID:35460690 A. gambiae salivary D7L1 and D7L3 proteins are vasodilators |
| GO:0090729 toxin activity | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: This annotation is an OVER-ANNOTATION. D7L3 does not meet the GO definition of toxin activity, which requires "initiating pathogenesis (leading to an abnormal, generally detrimental state)". D7L3 transiently modulates host physiology without causing disease or tissue damage - it is a kratagonist, not a toxin. Reason: The GO:0090729 definition states: "Interacting selectively with one or more biological molecules in another (target) organism, initiating pathogenesis (leading to an abnormal, generally detrimental state) in the target organism." D7L3 does NOT meet this definition because: (1) It does not initiate pathogenesis - it facilitates blood feeding, not disease; (2) It does not cause tissue damage - it transiently sequesters host serotonin; (3) The effect is reversible and temporary; (4) The host returns to normal after feeding. The UniProt keywords "Hemostasis impairing toxin" that led to this annotation via GO_REF:0000043 are technically inaccurate - anti-hemostatic activity through kratagonism is fundamentally different from toxin activity. A more accurate MF annotation is serotonin binding (GO:0051378). Supporting Evidence: UniProt:A0A1S4HE90 Modulates blood feeding of female mosquitoes on vertebrate species by binding and sequestering different mediators involved in the host response PMID:35460690 AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. |
| GO:0097746 blood vessel diameter maintenance | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: This annotation is reasonable but somewhat indirect. D7L3 affects blood vessel diameter by sequestering serotonin, preventing vasoconstriction. The annotation captures the physiological outcome. Reason: D7L3 does influence blood vessel diameter through its vasodilatory activity, as shown by PMID:35460690. However, this is not the core function - it is a downstream consequence of serotonin binding and sequestration. The core function is serotonin binding, which then prevents serotonin-induced vasoconstriction. This annotation is accurate but represents a secondary effect rather than the primary molecular function or biological process involvement. Supporting Evidence: PMID:35460690 A. gambiae salivary D7L1 and D7L3 proteins are vasodilators |
| GO:0051378 serotonin binding | IDA PMID:35460690 Novel salivary antihemostatic activities of long-form D7 pro... | NEW | Summary: D7L3 binds serotonin with high affinity as demonstrated by isothermal titration calorimetry. This is the core molecular function of D7L3 and is unusual for a long-form D7 protein. Reason: PMID:35460690 demonstrates serotonin binding by D7L3 using PEAQ-ITC: "AngaD7L3 binds serotonin" with thermodynamic parameters measured. This is the primary molecular function. PMID:35568118 confirms "The unusual D7L, D7L3, can also bind serotonin in the Cellia species An. gambiae" - highlighting that serotonin binding in a long-form D7 is unusual. UniProt states "Binds serotonin with high affinity (PubMed:35460690, PubMed:35568118)" [UniProt A0A1S4HE90]. Importantly, D7L3 is specific for serotonin among biogenic amines - it does NOT bind tryptamine, octopamine, dopamine, or adrenaline [UniProt A0A1S4HE90]. It only binds weakly to noradrenaline and histamine [PMID:35568118]. This makes serotonin binding (GO:0051378) the appropriate annotation, NOT histamine binding. Supporting Evidence: PMID:35460690 AngaD7L3 binds (E) serotonin (5-HT). Titration curves are representative of at least three measurements. PMID:35568118 The unusual D7L, D7L3, can also bind serotonin in the Cellia species An. gambiae. UniProt:A0A1S4HE90 Does not bind tryptamine, octopamine, dopamine, adrenaline, leukotriene C4, leukotriene D4, leukotriene B4, ADP and U-46619, a stable analog of thromboxane A2 |
| GO:1900047 negative regulation of hemostasis | IDA PMID:35460690 Novel salivary antihemostatic activities of long-form D7 pro... | NEW | Summary: D7L3 inhibits serotonin-induced platelet aggregation, which represents negative regulation of hemostasis. This is a core biological process annotation. Reason: PMID:35460690 demonstrates that "AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction." By sequestering serotonin (which promotes platelet aggregation), D7L3 negatively regulates the host hemostatic response. UniProt confirms "Inhibits agonist-induced platelet aggregation (PubMed:35460690)" [UniProt A0A1S4HE90]. GO:1900047 definition "Any process that stops, prevents or reduces the frequency, rate or extent of hemostasis" accurately captures this function. This is a more accurate BP annotation than the inferred "toxin activity". Supporting Evidence: PMID:35460690 AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction UniProt:A0A1S4HE90 Inhibits agonist-induced platelet aggregation |
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Download this section (compressed HTML)Q: Why did D7L3 evolve serotonin binding while other A. gambiae D7 long-forms (D7L1, D7L2) retained eicosanoid binding? What structural features enable this unusual specificity?
Suggested experts: Calvo E, Andersen JF, Ribeiro JMC
Q: What is the crystal structure of D7L3 bound to serotonin, and how does the binding pocket compare to short-form D7 proteins that also bind serotonin?
Suggested experts: Andersen JF, Garboczi DN
Experiment: Generate D7L3 knockout A. gambiae using CRISPR and measure blood meal size, feeding time, and feeding success rate compared to wild-type controls. Measure serotonin levels at the bite site in host tissue.
Hypothesis: D7L3 knockout mosquitoes will have reduced blood feeding efficiency due to host hemostatic responses, specifically serotonin-mediated platelet aggregation and vasoconstriction
Type: Gene knockout and feeding assay
Experiment: Solve the crystal structure of D7L3 in complex with serotonin and compare to known structures of Aedes D7L1 (which binds biogenic amines in its C-terminal domain) and Anopheles D7r4 (a short-form serotonin binder)
Hypothesis: D7L3's serotonin binding site is structurally distinct from the C-terminal biogenic amine site found in Aedes D7L1
Type: X-ray crystallography
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