Proc. Natl. Acad. Sci. USAVol. 91, pp. 138-142, January 1994Pharmacology

Sialokinin I and II: Vasodilatory tachykinins from the yellow fevermosquito Aedes aegyptiDONALD E. CHAMPAGNE* AND JOSE M. C. RIBEIRODepartment of Entomology, Center for Insect Science, University of Arizona, Tucson, AZ 85721

Communicated by John H. Law, August 19, 1993

ABSTRACT The saliva of the mosquito Aedes aegypti haspreviously been reported to contain a 1400-Da peptide withpharmacological properties typical of a tachykinin. In thepresent study this vasodilator has been purified to homogeneityand found to consist of two peptides: sialokinin I, with thesequence Asn-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2,and sialokinin II, identical to sialokinin I except for an Asp inposition 1. These peptides are present in amounts of 0.62 and0.16 pmol (711 and 178 ng), respectively, per salivary glandpair. When assayed on the guinea pig ileum, both peptides areas active as the mammalian tachykinin substance P, with Ko.svalues of 5.07, 6.58, and 4.94 nM for sialokinin I, sialokinin II,and substance P, respectively.

Blood-feeding insects are confronted with a number of ob-stacles in their quest for food. Even after a host has beenlocated, there remains the problem of finding a feeding site,as blood vessels occupy only a fraction of the skin area.Further, blood loss is defended against by an elaborate arrayof hemostatic mechanisms including vasoconstriction (1). Tocounter these defenses, hematophagous insects secrete intheir saliva a variety of substances including vasodilators (1,2). Besides helping to maintain blood flow during feeding,vasodilators increase the probability of finding blood (anddecrease probing times) by increasing the size of the targetvenules and arterioles.The yellow fever mosquito Aedes aegypti produces a 1400-

Da vasodilatory peptide characterized as a tachykinin, basedon its endothelium-dependent relaxation of aortic rings, con-traction of guinea pig ileum preparations, cross-desensitiza-tion with the vertebrate tachykinin substance P, and reactionwith an anti-substance P antibody (3). In the present report weexamine further the nature of this vasodilator.

EXPERIMENTAL PROCEDURESMaterials. Organic reagents, substance P, and histamine

were purchased from Sigma. Salts were of American Chem-ical Society standard or analytical grade. Synthetic peptideswere prepared by the Arizona Research Laboratories Divi-sion of Molecular Biology, using.a Gilson 422 automatedmultiple peptide synthesizer with fluoren-9-ylmethoxycar-bonyl (Fmoc) a protection and PyBOP (benzotriazol-l-yloxytrispyrrolidinophosphonium hexafluorophosphate;Nova Biochem) coupling activation. Cleavage with reagentKwas followed by precipitation and washing with t-butylmethyl ether, dissolution in 15-50%o HOAc, and direct ly-ophilization. Peptides were HPLC purified using a RaininHPLX system (Woburn, MA) with a C18 RP 21.4 x 250 mmDynamax column, using a gradient of 0-60%o acetonitrile/0.1% trifluoroacetic acid (TFA) at a flow rate of 16 ml/min.

Insects. A. aegypti Rockefeller strain (Diptera:Culicidae)mosquitoes were reared at 27°C under a 16-hr light/8-hr dark

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cycle and fed crushed guinea pig pellets. Adults were fed 10%(wt/vol) sucrose but not blood.

Isolation of Sialokinin. Salivary glands were dissected inphosphate-buffered saline from 3- to 10-day-old adult sugar-fed females. The glands were transferred to 10 mM Tris (pH8.0) and stored at -79°C in aliquots of 20 pairs per 20 ,ul ofbuffer in siliconized Eppendorf tubes until 1800 pairs hadbeen accumulated. As the salivary gland homogenates lackdetectable protease activity, protease inhibitors were notincluded in the phosphate-buffered saline or Tris buffers. Theglands were subsequently homogenized by holding the tubes(submerged in a 100-ml beaker of water) about 1 cm below theprobe tip of a Branson 450 sonicator, run with power setting7 and 40% duty cycle to allow dissipation of heat. Homog-enized glands were microcentrifuged for 5 min at 14,000 rpmand pooled in batches of 300-500 pairs, and the supematantwas fractionated through a 30-kDa Centricon (Amicon) filter.Each tube was rinsed with 50 ,ul of Tris buffer, and the rinseswere added to the Centricon tube for a second centrifugation.The filtrate was acidified to 8 mM HCI for reversed-phaseHPLC (RP-HPLC). The retentate was stored for subsequentprocessing of larger enzymes.RP-HPLC of the acidified filtrate was performed with a

Macrosphere 300 C18 5-,um 4.6 x 250 mm column (AlltechAssociates) using a Milton Roy 4000 (Rochester, NY) seriespump, detector, and integrator. Filtrate was loaded on thecolumn (two 5-ml injections) with 8 mM aqueous HCI (solu-tion A) and run for 10 min at 1.0 ml/min. A gradient of0-60%acetonitrile in 8 mM aqueous HCI (solution B) was developedover 60 min, followed by 10 min of isocratic solution B.Fractions were collected at 1-min intervals. After bioassay(see below), the active fractions were pooled and rechro-matographed using the same parameters but substituting0.1% TFA for the 8 mM HCl (4).

Synthetic peptides were separated by using the samecolumn and an isocratic solvent system of 100 mMNH4COOH/20% acetonitrile, pH 6.8, run at 0.5 ml/min. Forcomparison of the A. aegypti vasodilator to the syntheticpeptides, 100 salivary gland pairs were chromatographedusing this HPLC system, and fractions were collected everyminute for bioassay. For this experiment the injection loop,all HPLC tubing, and the syringe were replaced beforechromatographing the salivary gland homogenates to ensureagainst the possibility of contamination.

Bioassay. Aliquots (50 ,ul) of each fraction were combinedwith 10 ,ul of bovine serum albumin at 10 mg/ml, dried in aSpeed-Vac, and resuspended in 50 ,ul of pH 7.5 Tyrode'ssolution (3). Synthetic peptides were dissolved in 1 mMaqueous HCI at 1 mg/ml and serially diluted in Tyrode'ssolution for assay. In those experiments designed to identifythe particular peptide present in Aedes salivary glands, theentire fraction was dried for assay. Fractions and peptideswere tested with guinea pig terminal ileum preparations

Abbreviations: TFA, trifluoroacetic acid; RP-HPLC, reversed-phase HPLC; LTTK, locustatachykinin.*To whom reprint requests should be addressed.

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suspended in Tyrode's solution at 37°C, bubbled with 95%02/5% C02- Isotonic contractions against a load of 1.5 g weremeasured; to condition the ileum and assess its responsive-ness, the preparation was contracted with histamine at 200ng/ml, initially at 3-min intervals and subsequently afterevery three fractions. To prevent desensitization, the prep-aration was allowed to contract for 30 s before being rinsedwith fresh Tyrode's solution. Estimates of the tachykinincontent of each fraction were made by bracketing the sampleswith known doses of substance P. Dose-response curveswere constructed by bioassaying substance P and syntheticpeptides at 10-10, 3 x 10-9, 10-9, 3 x 10-8, 10-8, and 10-7 M.Measurements were performed in triplicate and analyzedusing the SAS Institute (Cory, NJ) GLM procedure.Amino-Terminal Sequencing. Edman degradation sequenc-

ing and electrospray mass spectrometry were performed byHarvard Microchemistry, under the direction of WilliamLane. Approximately 100 pmol of purified vasodilator wasapplied directly to a Polybrene precycled glass fiber filter ina reduced-volume microcartridge. The sample was subjectedto automated Edman degradation on an Applied Biosystemsmodel 477A protein sequencer using the manufacturer'srecommendations for faster cycle time (30 min) and thesensitivity enhancements described by Tempst and Riviere(5). The resultant phenylthiohydantoin amino acid fractionswere manually identified using an on-line Applied Biosys-tems model 120A HPLC and Shimadzu CR4A integrator.

Electrospray Ionization Mass Spectrometry. Mass spectrawere recorded on a Finnigan-MAT TSQ-700 (San Jose, CA)triple quadrapole mass spectrometer equipped with an elec-trospray ion source. The sample (2 pmol/pi) in 25% aceto-nitrile/0,1% TFA was directly infused at a flow rate of 2iA/min, while 2-methoxyethanol (2 1d/min) was provided asa sheathing solvent. The electrospray needle was operated ata voltage differential of -3.7 keV. Spectra were recorded byscanning the m/z range of 500-1800 in 3 s and averaging 32scans in the profile mode (6). The averaged scans were thensummed to produce the final data.

RESULTSThe A. aegypti salivary vasodilator was isolated by usingfractionation through a 30-kDa Centricon filter and tworeversed-phase HPLC steps. During the initial HPLC puri-fication, in which the homogenate was acidified with 8 mMHCl, the contractile activity eluted was associated with adouble peak (Fig. IA). The components of the peak wereresolved in a second HPLC step using 0.1% TFA as thepairing ion. Activity was associated with a single, apparentlyhomogeneous peak (Fig. 1B).Edman degradation of the purified vasodilator yielded the

sequence Asx-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Met. Theinitial residue was identified as Asn or Asp. Electrosprayionization MS gave a major M + H+ peak at 1145.4, a secondpeak at 1162.5 (presumably due to oxidation of the singlemethionine residue), and a smaller peak at 1031 (ascribed tocleavage of the amino-terminal residue). If Asn was the firstresidue, the unamidated peptide would have a mass of1145.41 (and so M + H+ = 1146.41); if the first residue wasAsp, the peptide would be 1146.41 Da (M + H+ = 1147.41).The discrepancy of 1 Da between the observed M + H+ peakand the calculated mass of 1146.4 Da for the unamidated Asn-peptide is consistent with amidation at the carboxyl terminus,a conclusion also supported by the biological activity datareported below. Much less abundant mass observed at m/z =1146.4 (with the corresponding sulfoxide at 1162.5) suggestedthat the additional presence of the Asp- peptide could not beexcluded.To distinguish the identity of the first amino acid, we

synthesized both possible forms of the peptide. However, we

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FIG. 1. HPLC purification and bioassay of 1800 A. aegyptisalivary gland pairs. (A) Initial RP-HPLC purification on a Macro-sphere 300 C18 7-,&m column: 0-10 min, 8 mM aqueous HCl (solventA); 10-70 min, 0-60%o acetonitrile/8 mM HCl (solvent B); 70-80min, isocratic solvent B. (B) Second RP-HPLC step, which wasidentical to the initial one except for the substitution of 0.1% TFA for8 mM HCI. Guinea pig ileum constricting activity, compared withmaximal contractions produced by histamine at 200 ng/ml, is alsoshown (o).

were unable to resolve the two synthetic peptides by anyHPLC system using HCl or TFA (pH 2.0) as pairing ions.Taking advantage of the difference in pKa of the amino andacid moieties of Asn and Asp, respectively, we were able toresolve the peptides with an isocratic mobile phase of 0.1 Mammonium formate/20% acetonitrile, pH 6.8 (Fig. 2C). Anal-ysis of 100 salivary gland pairs (Fig. 2A) indicated bioactivityeluting from the column coincident with both syntheticpeptides (Fig. 2B). A ratio of 4:1 of the Asn- to the Asp- formsof the peptide was observed (Fig. 2B). The failure of thepeptides to resolve with strongly acidic mobile phases ac-counts for the finding of a single biological activity peak in ourinitial HPLC strategy.Workup of the 1800 salivary gland pairs yielded 1.6,g of

combined tachykinins, or 889 pg (0.78 pmol) per pair, calcu-lated from the peak area and a calibration curve fromsubstance P. The activity of freshly prepared salivary glandhomogenates was equivalent to 1.2 ng of substance P per pair,or 972 pg of the synthetic peptides (based on the comparativebiological activities discussed below), giving an approximateyield of 91%.The activity of the synthetic peptides was compared with

that of the mammalian tachykinin substance Pin a guinea pigileum bioassay. Both peptides had very similar potencies tosubstance P in this assay, with KO.5 values of 6.58 (95%confidence interval: 5.22-7.94) nM for the Asn- derivative,5.07 (4.32-5.82) nM for the Asp- form, and 4.94 (3.33-6.55)nM for substance P (Fig. 3). There is some indication that themosquito tachykinins produce stronger contractions at con-centrations of 3 x 10-8 M and above, but the physiologicalrelevance of this is uncertain.

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140 Pharmacology: Champagne and Ribeiro

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FiG. 2. Confirmation of sialokinins I and II in A. aegypti salivaryglands. (A) One hundred A. aegypti salivary gland pairs werechromatographed on a Macrosphere 300 Cls 5-,um column with anisocratic mobile phase of 20%o acetonitrile/0.1 M ammonium for-mate, pH 6.8. (B) Results of a guinea pig ileum assay using 1-minfractions from A. SP, substance P. (C) Synthetic sialokinins I (rightpeak) and II (left peak) were separated using the same protocol.

DISCUSSION

The A. aegypti vasodilator was previously found to cross-desensitize with the mammalian tachykinin substance P andto bind an anti-substance P antibody (3). In this study we havepurified and sequenced the vasodilator and found it to consistoftwo tachykinins, which we name sialokinins from the greek"sialos" for saliva. The more abundant peptide, with Asn asthe amino-terminal residue, we call sialokinin I; the Asp-analogue is named sialokinin II. Both peptides contain thecarboxyl-terminal sequence Phe-Xaa-Gly-Leu-Met-NH2,which characterizes all members ofthe tachykinin family andis required for binding to tachykinin-specific receptors (7).The Lys at position 5 accounts for the previously observedsensitivity to trypsin and chymotrypsin (3). Overall sequenceidentity with previously described vertebrate tachykinins ishigh, ranging from a minimum of 4 to a maximum of 7 (out of10) amino acids (Fig. 4). The sialokinins are most similar toscyliorhinin, isolated from the dogfish shark (9), but they alsoresemble a variety of amphibian tachykinins including hy-lambatin and physaelamin. The sialokinins appear to be more

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FiG. 3. Dose-response curves of substance P (SP), syntheticsialokinin I (SK1), and synthetic sialokinin II (SK2) tested on aguinea pig ileum. The ileum was suspended in Tyrode's solution (pH7.5) gassed with 95% 02/5% C02, and isotonic contractions weremeasured against a 1.5-g load. Each point shows the mean ± SE ofthree replicates.

similar to some vertebrate tachykinins than they are to theonly other known invertebrate tachykinin, eledoisin (10).Munekata et al. (11), in a study of carboxyl-terminal

heptapeptides (I-II-Phe-III-Gly-Leu-Met-NH2), found thattwo classes of tachykinins could be defined: those with Glnor Asn in position I and an aromatic amino acid in position III,related to substance P, and those with Asp in position I andan aliphatic amino acid (Val or Ile) in position III, related toneurokinin A and B. Substance P and related compoundsinteract with the NK1 receptor, present in the guinea pigileum among other tissues, and neurokinin-like peptidesinteract with NK2 and NK3 receptors, found for example inrat duodenum. The sialokinins mix characters of both tachy-kinin types, having an Asp at position I and a Tyr at positionIII. The model peptide results predict that the sialokininsshould be less active than substance P on the guinea pigileum, but this was not observed to be the case. AlthoughMunekata et al. (11) concluded that the identity ofamino acidII was not important, the basic character of Lys in positionII may compensate for the acidic Asp in position I, producinga more substance P-like carboxyl-terminal region with higheraffinity for NK1 receptors.Tachykinins produce a variety of effects in addition to

smooth muscle contraction and endothelium-dependent di-lation. Substance P causes the release of histamine from mastcells and enhances human neutrophil phagocytosis, but theseeffects are dependent on the sequence of basic amino acids(Arg-Pro-Lys-Pro) at the amino-terminal end and requiremicromolar concentrations in vitro (12-17) or 100 nM con-centrations in vivo (18). Some tachykinins that lack thissequence (possibly including the sialokinins) can inhibit theeffect, because the carboxyl-terminal sequence is also in-volved in binding to a receptor on mast cells. On the otherhand, substance P and neurokinins can activate macrophagesat low concentrations (0.1-1.3 nM), concentrations compa-rable to those required for smooth muscle contraction (19,20). As this activity is dependent on the carboxyl-terminalsequence, the sialokinins may also activate macrophages atthe site of feeding. This effect may be particularly significantin light of the role of A. aegypti as a vector of a large arrayof disease-causing parasites. As the infectivity of someviruses including Denge is enhanced by antibody-dependentmechanisms (21), we can speculate that the evolutionaryselection of tachykinins as salivary vasodilators in A. aegypti

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Aedes aegyptiSialokinin ISialokinin II

MammalsSubstance PNeurokinin ANeurokinin BNeuropeptide K

Neuropeptide y

Asn-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Net-NH2Asp-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Ket-NH2

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LOU-KOt-NH2His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-XMt-NH2Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NI2Asp-Ala-Asp-Ser-Ser-Ile-Glu-Lys-Gln-Val-Ile-

Ser-His-Lys-Arg-Leu-Tyr-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Net-NH2

Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-MOt-NH2

AmphibiansPhysglaleWin pGlp-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2[Lys ,Thr ]Physalalemin

pGlp-Ala-Asp-Pro-Lys-Thr-Phe-Tyr-Gly-Leu-Met-N12Uperolein pGlp-Pro-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2Phyllomedusin Glp-----Asn-Pro-Asn-Arg-Phe-Ile-Gly-Leu-Net-NH2Kassinin Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH2Hylambatin Asp-Pro-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Gly-Leu-Met-NH2

FishScyliorhinin Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2

Mollusc

Eledoisin Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2

LocustatachykininsLTTK ILTTK IILTTK IIILTTK IV

Gly-Pro-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2Ala-Pro-Leu-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2

FIG. 4. Comparison of the sequence of sialokinins with selected tachykinins and locustatachykinins (LTTKs). Peptide sequences are fromrefs. 7 and 8. Amino acid residues common to the sialokinins and other tachykinins are in boldface type.

may have consequences for the vectorial capacity of thisinsect.The habit offeeding on blood has arisen many times among

the arthropods; each independent arrival at hematophagyrequires solutions to similar problems posed by the hemo-static defenses of the vertebrate hosts. While this has pro-duced convergent traits in terms of the pharmacologicalactivities of the arthropod's saliva (vasodilators, antiplateletfactors), at the molecular level a wide variety of compoundshave been employed. The sandfly Lutzomyia longipalpususes a unique peptide, maxadillian, as a vasodilator (22), arole filled by nitrosylheme proteins in Rhodnius prolixus (23,24), prostaglandins in ticks (25), a peroxidase in Anophelesalbimanus (26), and tachykinins in A. aegypti. It seems mostparsimonious to hypothesize that these compounds werealready present in some "domestic" function and wereselectively overexpressed and/or structurally modified overevolutionary time because of their fortuitous effects onvertebrate physiology. Salivary glands may therefore providea window on biochemical processes that otherwise occur atlow levels or only in a few specialized cells. Indeed, immu-noreactivity to an anti-substance P antibody has been re-ported in a variety of invertebrates, including dipterans(27-29), locusts (30), lepidopterans (31), several cockroachspecies (32-34), crustaceans (35), and even the primitivecoelenterate Hydra (36), where substance P also stimulateshead regeneration (37). Additionally, Li et al. (8) haveisolated from Drosophila melanogaster a cDNA clone with40-48% amino acid identity (within transmembrane regions)to mammalian tachykinin receptors. When expressed inXenopus oocytes, the gene product responds as an NK1receptor. The presence ofa developmentally regulated tachy-

kinin receptor in Drosophila suggests that this class ofneuropeptides is functionally significant in insects.Four LTTKs, isolated from the nervous system ofLocusta

migratoria, have been described as tachykinins (38-40), butthese lack the signature carboxyl-terminal sequence of allvertebrate tachykinins and rather display sequence similarityto the central and amino-terminal regions of some vertebratetachykinins (Fig. 4). These peptides have not been shown tobe recognized by anti-substance P antibodies or to cross-desensitize insect tissues to substance P, but they produceeffects in insect smooth muscle similar to the effects oftachykinins on vertebrate tissues. A second Drosophila re-ceptor, which has 38% sequence identity with vertebrateNK3 receptors, responds to LTTK II (but not LTTK I) and,to a lesser extent, substance P and physaelemin (41). It seemsbest to consider LTTKs as members of a distinct neuropep-tide family, with an as yet inconclusive relationship to thetachykinins.Although several lines of evidence indicate the widespread

presence of tachykinins as invertebrate neuromodulators, itshould also be noted that the most significant aspect of thesequence similarity of sialokinins to vertebrate tachykininsinvolves only six amino acids. A sequence ofthis length couldarise through convergent evolution, in which case the sialo-kinins would be analogues and not homologues of the ver-tebrate peptides. It will be necessary to determine the se-quence and structure of the gene(s) encoding the sialokininsto settle this question. The same approach will also berequired to resolve the relationship of sialokinin I and II: arethey products of separate genes, or are they allelic variantsthat have arisen from a single base pair change?

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The discovery of two tachykinins in A. aegypti salivaryglands emphasizes once again the diversity of pharmacolog-ically active compounds to be found in these organs; theircontinued study is certain to prove profitable.

We thank Dr. William Lane of Harvard Microchemistry forperforming the mass spectrometry and Edman sequence analysis.Dr. Jan Veenstra provided timely advice on the HPLC strategy.Roberto Nussenzveig is thanked for his technical assistance. Thepaper benefited from critical reading by Dr. Jan Veenstra and Dr.Herman Lehman. This work was supported by National Institutes ofHealth Grant 18694 to J.M.C.R.

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Proc. Natl. Acad. Sci. USA 91 (1994)

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