- Email: [email protected]
The neuropeptidomics of Ixodes scapularis synganglion
J O U R NA L OF PR O TE O MI CS 72 ( 20 0 9 ) 1 0 4 0–1 0 4 5
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / j p r o t
The neuropeptidomics of Ixodes scapularis synganglion Susanne Neupert a,c,⁎, William K. Russell b , Reinhard Predel c , David H. Russell b , Otto F. Strey d , Pete D. Teel d , Ronald J. Nachman a,⁎ a
Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USA The Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA c Institute of Zoology, Friedrich-Schiller-University, Erbertstrasse 1, Jena, 07743, Germany d Department of Entomology, Texas A&M University, College Station, TX 77843, USA b
AR TIC LE I N FO
ABS TR ACT
Article history:
Ticks (Ixodoidea) likely transmit the greatest variety of human and animal pathogens of any
Received 8 May 2009
arthropod vector. Despite their medical significance little data is available about the
Accepted 10 June 2009
messenger molecules in the central nervous system that coordinate all physiological processes in these animals, including behaviour. In our study, we performed the first
Keywords:
comprehensive neuropeptidomic analysis of a tick species by using MALDI-TOF mass
Tick neuropeptidomics
spectrometry. Specifically we analyzed the neuropeptides in the synganglion of Ixodes
Ixodes
scapularis. The forthcoming sequence of the genome of this species will represent the first
Amblyomma
genomic analysis of a member of the large subphylum Chelicerata. For our approach we
Acari
used information from predicted neuropeptide precursor sequences found in EST databases
MALDI-TOF mass spectrometry
[Christie, AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174–185] as well as data obtained by complete de novo sequencing. The direct tissue profiling yielded 20 neuropeptides from 12 neuropeptide precursors. The sequences of these neuropeptides are not as unique as predicted; a comparison with the peptidome of other invertebrates shows a close relationship with insect neuropeptides. This work will provide a resource for studying tick neurobiology and will hopefully also help to identify novel targets for tick and tick-borne disease control. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
As obligate blood feeders, ticks (Ixodoidea) transmit a great variety of human and animal pathogens, including Lyme disease/borreliosis (transmitted by e.g. Ixodes ricinus in Europe and Ixodes scapularis in North America), tick-borne meningoencephalitis, Rocky Mountain spotted fever, cattle fever, and other serious diseases [2,3]. Despite their medical and economic significance only sporadic data are available about
messenger molecules in the central nervous system that coordinate the physiological processes in these animals, including behaviour. In particular, the neuropeptides which constitute the most diverse group of messenger molecules are poorly known from ticks and related arachnids [4]. Only a single CAPA-peptide [5] and a myoinhibitory peptide [6] were biochemically identified from the synganglion; other peptides that were isolated from Amblyomma testudinarium [7] and Ixodes sinensis [8] have still to be confirmed as genuine tick
⁎ Corresponding authors. Neupert is to be contacted at Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USA. Tel.: +1 3641 9 49191; fax: +1 3641 9 49192. Nachman, tel.: +1 979 260 9315; fax: +1 979 260 9377. E-mail addresses: [email protected] (S. Neupert), [email protected] (R.J. Nachman). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.06.007
J O U R NA L OF PR O TE O MI CS 7 2 ( 2 00 9 ) 1 0 4 0–1 0 4 5
neuropeptides. This lack of information is in contrast to information on neuropeptides of insects for which numerous neuropeptide families have been described and for which there are many completed and ongoing genome sequencing projects. On the other hand, the neuroanatomy of the neuroendocrine system of ticks was recently described in detail [9], and the availability of substantial ESTs for different tick species generated a source for the identification of putative neuropeptides of ticks (see [1]). In addition, the ongoing Ixodes scapularis Genome Project, the first to sequence a tick (and chelicerate) genome, will provide an unparalleled resource for studying tick biology [10]. The genome information will also help to identify novel targets for tick and tickborne disease control. In our study, we used I. scapularis to perform the first comprehensive neuropeptidomic analysis of any tick species by using MALDI-TOF mass spectrometry. Specifically we analyzed the neuropeptides in the central nervous system (synganglion) to close the gap between the numerous predictable neuropeptides and the neuropeptides which are indeed expressed in the nervous system. Since predictions of neuropeptide cleavage from precursor proteins are not always correct, such biochemical identification is essential for the design of future experiments; particularly in taxa that have never been analyzed before.
2.
Material and methods
2.1.
Animals
1041
Ixodes scapularis was obtained from Insect Service GmbH (Berlin, Germany). Nymphs of the Gulf Coast tick Amblyomma maculatum from a colony originating from ticks collected in Refugio County, TX were fed on poultry at the Tick Research Laboratory (Department of Entomology, Texas A&M University, College Station) and maintained at 23 °C, 85 RH, and a 15 h photophase. Following ecdysis, adult ticks were transferred to the Areawide Pest Management Research Station, Southern Plains Agricultural Research Center (USDA-ARS, College Station, TX), where all preparations on these ticks were performed. Only unfed adult ticks were used throughout the experiments.
2.2. Dissection and sample preparation for mass spectrometry (Fig. 1) Adult ticks (without blood meal; n = 20) were fixed with needles and the body cavity opened with a scissor. The fused central nervous system (synganglion) was dissected and placed in a
Fig. 1 – Dissection of the nervous system for neuropeptide profiling. A: Localisation of the central nervous system (synganglion; SG) within the tick body, scale bar = 600 µm; B: pieces of nervous tissues which were used for direct tissue profiling by means of MALDI-TOF MS; C: overview of the sample preparation for mass spectrometry.
1042
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separate chamber filled with insect saline (pH 7.25) of the following composition: NaCl (7.50 g/l), KCl (0.20 g/l), CaCl2 (0.20 g/l) and NaHCO3 (0.10 g/l); a solution that is isotonic with the hemolymph of ticks and prevents cell disruption during the dissection. Using fine scissors, synganglia (600– 650 µm) were cut in small pieces and transferred with a stainless steel insect pin into a drop of distilled water on the sample plate for MALDI-TOF mass spectrometry. The water was subsequently removed using a glass capillary. Approximately 50 nl of matrix solution (saturated α-cyano-4-hydroxycinnamic acid dissolved in methanol/water [1:1]) was pumped onto the dried tissue over a period of about 5 s using a Nanoliter injector (World Precision Instruments, Berlin, Germany). Each preparation was air-dried and covered with pure water for a few seconds, which was removed by cellulose paper.
2.3.
MALDI-TOF mass spectrometry
MALDI-TOF analyses were performed on an ABI 4800 proteomics analyzer (Applied Biosystems, Framingham, MA). Due to the nature of the samples all acquisitions were taken in manual mode. Initially the instrument was operated in reflectron mode, in order to determine the parent masses. A laser intensity of 3800 was typically employed for ionizing the neuropeptides. For the tandem MS experiments (performed in gas on and gas off mode, respectively), the CID acceleration was 1 kV in all cases. The number of laser shots used to obtain a spectrum varied from 600 to 4000, depending on signal quality. The fragmentation patterns were used to manually determine the sequence of the peptides by using the Data ExplorerT software package.
3.
Results and discussion
Initially, synganglia of I. scapularis were dissected, the ganglionic sheath removed and these preparations used for MALDI-TOF mass spectrometry. The resulting mass spectra revealed only a few ion signals and the ion intensity was generally low. For that reason, synganglia were cut in smaller pieces of approx. 100 µm. These pieces were then directly transferred to a sample plate for MALDI-TOF mass spectrometry (see Fig. 1). Only mass spectra from such preparations yielded ion intensities that were sufficient for tandem mass spectrometry (Fig. 2A). In the mass range typical of shorter neuropeptides (600–3500 Da), numerous putative neuropeptides were detectable (Table 1). All prominent ions were fragmented by tandem mass spectrometry using the same samples that were analyzed for mass fingerprints before (see Fig. 3). Using the information from ESTs (see [1]), we could assign a large number of ion signals to predicted neuropeptides (see Fig. 2B,C), including RFamides (sulfakinins, short neuropeptide F), diuretic hormone (DH-35), corazonin, orcokinins, CAPA-periviscerokinin, SIFamides, tachykininrelated peptides (TKRP), and proctolin. In addition, the recently predicted allatostatin-C [11] and myoinhibitory peptide (MIP) [6]; were found to be expressed. Nearly all peptides which were observed in our mass spectra belong to neuropeptide families that are known to activate specific receptors in
Fig. 2 – Representative MALDI-TOF mass spectrum of a piece of tissue from the synganglion of I. scapularis. A: Mass range of 600–3500 Da. B: Detail of A, which illustrates the high number of ion signals in the mass range of 600–1800 Da. C: Detail of A which shows the ion signal of the diuretic hormone (DH-35). All prominent ion signals were analyzed by tandem mass spectrometry, successfully assigned neuropeptides are labelled. SK: sulfakinin; sNPF: short neuropeptide F; cor: corazonin; Orc: orcokinin; PK: pyrokinin; PVK: periviscerokinin; Proc: proctolin; TKRP: tachykinin-related peptide; MIP: myoinhibitory peptide.
insects (see [12]). The numerous additional neuropeptides that were predicted in Christie (2008) from the same precursors were not found [1]. Altogether, 20 neuropeptides from 12 neuropeptide families have been detected by this first comprehensive neuropeptidomic approach on ticks. More neuropeptides such as insect kinin-related peptides can be expected since the putative kinin receptor is already identified [13]. The prediction that the neuropeptidome of these arthropods is highly unique in comparison to other sequenced invertebrate genomes could not be confirmed. Indeed, the sequences of the tick neuropeptides show a close relationship with insect neuropeptides (e.g. Periplaneta americana, Drosophila melanogaster, Apis mellifera) although the evolutionary distance between ticks and insects is considerable. The class Insecta belongs phylogenetically to the subphylum Mandibulata and is closely related with Crustacea. The ticks belong to
1043
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Table 1 – Summary of the neuropeptides that were detected in the CNS of I. scapularis by MALDI-TOF MS. Peptide name Corazonin Diuretic hormone-35 sNPF sNPF3-11 Sulfakinin-1 Sulfakinin-2 Orcokinin-2 Orcokinin-3 Orcokinin-PP proctolin Periviscerokinin Pyrokinin-1 Pyrokinin-2 Pyrokinin-3 SIFamide TKRP-1 (2x) TKRP-2 Allatostatin-C Myoinhibitory peptide Pyrokinin-4
Peptide sequence
[M + H]+
MS
MS/MS
EST hits
pQTFQYSRGWTNa AGGLLDFGLSRGASGAEAAKARLGLKLANDPYGPa GGRSPSLRLRFa SPSLRLRFa QDDDYGHMRFa SDDYGHMRFa WYGHGDFDEIDNVGWPGFT-OH NFDEIDRTGFEGFY-OH AANAAAAPAAREDA-OH RYLPT-OH pQGLIPFPRVa RSNNFTPRIa GSFVPRLa GSFTPRIa AYRKPPFNGSIFa AFHAMRa GSGFFGMRa SGWKQCSFNAVSCFa ASDWNRLSGMWa TPFTPRLa
1369.63 3312.78 1244.73 974.58 1282.50 1126.47 1623.67* 1709.74 1269.62 649.36 1008.59 1103.61 774.46 776.44 1395.75 731.38 857.41 1562.68 1321.61 830.54
x x x x x x x x x x x x x x x x x x x x
x x x x x x – x x x x x x x x x x – x x
EL516967 EW915260 EW855501 EW941557 EW941557 EW865036
EW933575
EW859993 EL516783
sNPF, short neuropeptide F; PP, precursor peptide; TKRP, tachykinin-related peptide. *[M + Na]+ and [M + K]+ adduct ions only.
the subphylum Chelicerata (together with e.g. spiders, scorpions, and mites) which separated from the Mandibulata nearly 500 million years ago [14] and it comes therefore as a surprise that the neuropeptides of these only distantly related arthropods are that similar. Certainly, the similarity of neuropeptide sequences within the large phylum Arthropoda (with more than 1 million species) will make it quite difficult to identify novel targets for tick and tick-borne disease control by developing specific peptide mimetics. An interesting phenomenon is the sequence of a peptide precursor that contains the CAPA-periviscerokinin (PVK). In insects, the capa-gene usually contains several PVKs and a single pyrokinin with the C-terminal WFGPRLa [15]. In
+
Fig. 3 – CID mass spectrum of the peptide at [M + H] : 649.4 Da under condition of high gas. The fragments were analyzed manually and the resulting sequence is given in the inset. The y-, a- and b-type ions, which verified the amino acid sequence of proctolin, are labelled.
addition, a second gene always encodes for other FXPRLamides (pyrokinins, pheromone biosynthesis activating neuropeptides [16]. It seems possible that the capa-gene and the pyrokinin gene of insects evolved from a common ancestor gene, at least the relationship of their specific receptors suggests so [17–19]. The sequence-related tick precursor contains a PVK typical of the capa-gene of insects and three pyrokinins typical of the fxprl-gene. It can thus be speculated, that this gene is an ancestor of the two insect genes and the differentiation in capa and fxprl-genes occurred after the separation of arthropods from the other taxa. However, by de
Fig. 4 – CID mass spectrum of the substance at [M + H]+: 830.5 Da. The y-, a- and b-type ions, which were used to identify the amino acid sequence of a pyrokinin (PK-4), are labelled. The deduced sequence of TPFTPRL-NH2 could not be assigned to any of the existing tick ESTs but shows a very close relationship to insect pyrokinins (see Table 2).
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J O U R NA L OF PR O TE O MI CS 72 ( 20 0 9 ) 1 0 4 0–1 0 4 5
Table 2 – Amino acid sequences of Ixodes pyrokinin-4 and sequence-related FXPRLamides from different insect taxa. Species Lepisma saccharina Periplaneta americana Nezara viridula Tribolium castaneum Drosophila melanogaster Apis mellifera Ixodes scapularis
Peptide sequence
References
SPPFAPRLa SPPFAPRLa SPPFAPRLa SPPFAPRLa SVPFKPRLa IYLPLFASPLa TPFTPRLa
Unpublished [20] [21] [19] [17] [22] This study
Acknowledgments The authors wish to thank Allison Strey for technical assistance (College Station, TX). This study was supported by a grant from Deutsche Forschungsgemeinschaft (Predel 595/6-4), and a grant from the USDA/DOD DWFP Initiative (#0500-32000-001-01R) (RJN).
REFERENCES novo sequencing (Fig. 4), we found a tick pyrokinin (TPFTPRLa) which is not encoded on the above-mentioned capa/pyrokinin gene. The sequence of this pyrokinin is very closely related to the most conserved pyrokinin of insects (see Table 2) and suggests the occurrence of a second fxprl-gene in ticks that has not yet been predicted. The peptidomics approach which was performed on I. scapularis, was subsequently repeated with another tick species, Amblyomma americanum. The resulting mass spectra verified a very close relationship of these ticks, and most neuropeptides were identical in both species (see Fig. 5). We found no trace of opioid peptides that were isolated as abundant peptides from synganglia of other tick species [7,8]. Possibly, these peptides were indeed contaminations from mammalian blood, and the intestine of ticks crosses the synganglion. The data presented here will provide a basic resource for studying tick biology. The differentiation between predicted and biochemically identified neuropeptides will help to avoid experiments with putative peptides with uncertain biological relevance.
[1] Christie AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174–85. [2] Kaiser R. Tick-borne encephalitis. Infect Dis Clin North Am 2008;22:561–75. [3] Tickborne Diseases—National Center for Infectious Diseases (CDC). http://www.cdc.gov/ticks/diseases/. [4] Lees K, Bowman AS. Tick neurobiology: recent advances and the post-genomic era. Invert Neurosci 2007;4:183–98. [5] Neupert S, Predel R, Russell WK, Davies R, Pietrantonio PV, Nachman RJ. Identification of tick periviscerokinin, the first neurohormone of Ixodidae: single cell analysis by means of MALDI-TOF/TOF mass spectrometry. Biochem Biophys Res Commun 2005;338:1860–4. [6] Simo L, Park Y. Myoinhibitory peptide and pigment dispersing factor peptidergic inervation of the salivary glands of the blacklegged tick (Ixodes scapularis Say. The 2008 ESA Annual Meeting, November 16–19, 2008. [7] Liang JG, Zhang J, Lai R, Rees HH. An opioid peptide from synganglia of the tick, Amblyomma testindinarium. Peptides 2005;26:603–6. [8] Li J, Lui T, Yang H, Xu X, Lui Z, Lai R. Two neuropeptides from synganglia of the hard tick, Ixodes sinensis (Acari: Ixodidae). Chin Sci Bull 2006;51:2743–7.
Fig. 5 – Comparison of MALDI-TOF mass spectra of preparations from synganglia of I. scapularis and A. americanum (mass range 700–1500 Da). Most neuropeptides are identical in both species; the sequences of the modified Amblyomma PK-1, and 2 are RSNTFTPRI-NH2 and STPVPRL-NH2.
J O U R NA L OF PR O TE O MI CS 7 2 ( 2 00 9 ) 1 0 4 0–1 0 4 5
[9] Simo L, Slovák M, Park Y, Zitnan D. Identification of a complex peptidergic neuroendocrine network in the hard tick, Rhipicephalus appendiculatus. Cell Tissue Res 2009;335:639–55. [10] Pagel Van Zee J, Geraci NS, Guerrero FD, Wikel SK, Stuart JJ, Nene VM, et al. Tick genomics: the Ixodes genome project and beyond. Int J Parasitol 2007;37:1297–305. [11] Veenstra JA. Allatostatin C and its paralog allatostatin double C: the arthropod somatostatins. Insect Biochem Mol Biol 2009;39:161–70. [12] Hauser F, Cazzamali G, Williamson M, Blenau W, Grimmelikhuijzen CJ. A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera. Prog Neurobiol 2006;80:1–19. [13] Holmes SP, Barhoumi R, Nachman RJ, Pietrantonio PV. Functional analysis of a G protein-coupled receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor. Insect Mol Biol 2003;12:27–38. [14] Douzery EJ, Snell EA, Bapteste E, Delsuc F, Philippe H. The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils? Proc Natl Acad Sci U S A 2004;101:15386–91. [15] Predel R, Wegener C. Biology of the CAPA peptides in insects. Cell Mol Life Sci 2006;63:2477–90. [16] Predel R, Nachman RJ. The FXPRLamide (pyrokinin/PBAN) peptide family. In: Kastin AJ, editor. The handbook of biologically active peptides. San Diego: Academic Press/Elsevier Inc.; 2006. p. 207–12.
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[17] Hewes RS, Taghert PH. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 2001;11:1126–42. [18] Hauser F, Cazzamali G, Williamson M, Park Y, Li B, Tanaka Y, et al. A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum. Front Neuroendocrinol 2008;29:142–65. [19] Li B, Predel R, Neupert S, Hauser F, Tanaka Y, Cazzamali G, et al. Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum. Genome Res 2008;18:113–22. [20] Predel R, Kellner R, Kaufmann R, Penzlin H, Gäde G. Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach. Peptides 1997;18:473–8. [21] Predel R, Russell WK, Russell DH, Lopez J, Esquivel J, Nachman RJ. Comparative peptidomics of four related hemipteran species: pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins. Peptides 2008;29:162–7. [22] Hummon AB, Richmond TA, Verleyen P, Baggerman G, Huybrechts J, Ewing MA, Vierstraete E, Rodriguez-Zas SL, Schoofs L, Robinson GE, Sweedler JV. From the genome to the proteome: uncovering peptides in the Apis brain. Science 2006;314:647–9.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / j p r o t
The neuropeptidomics of Ixodes scapularis synganglion Susanne Neupert a,c,⁎, William K. Russell b , Reinhard Predel c , David H. Russell b , Otto F. Strey d , Pete D. Teel d , Ronald J. Nachman a,⁎ a
Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USA The Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA c Institute of Zoology, Friedrich-Schiller-University, Erbertstrasse 1, Jena, 07743, Germany d Department of Entomology, Texas A&M University, College Station, TX 77843, USA b
AR TIC LE I N FO
ABS TR ACT
Article history:
Ticks (Ixodoidea) likely transmit the greatest variety of human and animal pathogens of any
Received 8 May 2009
arthropod vector. Despite their medical significance little data is available about the
Accepted 10 June 2009
messenger molecules in the central nervous system that coordinate all physiological processes in these animals, including behaviour. In our study, we performed the first
Keywords:
comprehensive neuropeptidomic analysis of a tick species by using MALDI-TOF mass
Tick neuropeptidomics
spectrometry. Specifically we analyzed the neuropeptides in the synganglion of Ixodes
Ixodes
scapularis. The forthcoming sequence of the genome of this species will represent the first
Amblyomma
genomic analysis of a member of the large subphylum Chelicerata. For our approach we
Acari
used information from predicted neuropeptide precursor sequences found in EST databases
MALDI-TOF mass spectrometry
[Christie, AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174–185] as well as data obtained by complete de novo sequencing. The direct tissue profiling yielded 20 neuropeptides from 12 neuropeptide precursors. The sequences of these neuropeptides are not as unique as predicted; a comparison with the peptidome of other invertebrates shows a close relationship with insect neuropeptides. This work will provide a resource for studying tick neurobiology and will hopefully also help to identify novel targets for tick and tick-borne disease control. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
As obligate blood feeders, ticks (Ixodoidea) transmit a great variety of human and animal pathogens, including Lyme disease/borreliosis (transmitted by e.g. Ixodes ricinus in Europe and Ixodes scapularis in North America), tick-borne meningoencephalitis, Rocky Mountain spotted fever, cattle fever, and other serious diseases [2,3]. Despite their medical and economic significance only sporadic data are available about
messenger molecules in the central nervous system that coordinate the physiological processes in these animals, including behaviour. In particular, the neuropeptides which constitute the most diverse group of messenger molecules are poorly known from ticks and related arachnids [4]. Only a single CAPA-peptide [5] and a myoinhibitory peptide [6] were biochemically identified from the synganglion; other peptides that were isolated from Amblyomma testudinarium [7] and Ixodes sinensis [8] have still to be confirmed as genuine tick
⁎ Corresponding authors. Neupert is to be contacted at Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USA. Tel.: +1 3641 9 49191; fax: +1 3641 9 49192. Nachman, tel.: +1 979 260 9315; fax: +1 979 260 9377. E-mail addresses: [email protected] (S. Neupert), [email protected] (R.J. Nachman). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.06.007
J O U R NA L OF PR O TE O MI CS 7 2 ( 2 00 9 ) 1 0 4 0–1 0 4 5
neuropeptides. This lack of information is in contrast to information on neuropeptides of insects for which numerous neuropeptide families have been described and for which there are many completed and ongoing genome sequencing projects. On the other hand, the neuroanatomy of the neuroendocrine system of ticks was recently described in detail [9], and the availability of substantial ESTs for different tick species generated a source for the identification of putative neuropeptides of ticks (see [1]). In addition, the ongoing Ixodes scapularis Genome Project, the first to sequence a tick (and chelicerate) genome, will provide an unparalleled resource for studying tick biology [10]. The genome information will also help to identify novel targets for tick and tickborne disease control. In our study, we used I. scapularis to perform the first comprehensive neuropeptidomic analysis of any tick species by using MALDI-TOF mass spectrometry. Specifically we analyzed the neuropeptides in the central nervous system (synganglion) to close the gap between the numerous predictable neuropeptides and the neuropeptides which are indeed expressed in the nervous system. Since predictions of neuropeptide cleavage from precursor proteins are not always correct, such biochemical identification is essential for the design of future experiments; particularly in taxa that have never been analyzed before.
2.
Material and methods
2.1.
Animals
1041
Ixodes scapularis was obtained from Insect Service GmbH (Berlin, Germany). Nymphs of the Gulf Coast tick Amblyomma maculatum from a colony originating from ticks collected in Refugio County, TX were fed on poultry at the Tick Research Laboratory (Department of Entomology, Texas A&M University, College Station) and maintained at 23 °C, 85 RH, and a 15 h photophase. Following ecdysis, adult ticks were transferred to the Areawide Pest Management Research Station, Southern Plains Agricultural Research Center (USDA-ARS, College Station, TX), where all preparations on these ticks were performed. Only unfed adult ticks were used throughout the experiments.
2.2. Dissection and sample preparation for mass spectrometry (Fig. 1) Adult ticks (without blood meal; n = 20) were fixed with needles and the body cavity opened with a scissor. The fused central nervous system (synganglion) was dissected and placed in a
Fig. 1 – Dissection of the nervous system for neuropeptide profiling. A: Localisation of the central nervous system (synganglion; SG) within the tick body, scale bar = 600 µm; B: pieces of nervous tissues which were used for direct tissue profiling by means of MALDI-TOF MS; C: overview of the sample preparation for mass spectrometry.
1042
J O U R NA L OF PR O TE O MI CS 72 ( 20 0 9 ) 1 0 4 0–1 0 4 5
separate chamber filled with insect saline (pH 7.25) of the following composition: NaCl (7.50 g/l), KCl (0.20 g/l), CaCl2 (0.20 g/l) and NaHCO3 (0.10 g/l); a solution that is isotonic with the hemolymph of ticks and prevents cell disruption during the dissection. Using fine scissors, synganglia (600– 650 µm) were cut in small pieces and transferred with a stainless steel insect pin into a drop of distilled water on the sample plate for MALDI-TOF mass spectrometry. The water was subsequently removed using a glass capillary. Approximately 50 nl of matrix solution (saturated α-cyano-4-hydroxycinnamic acid dissolved in methanol/water [1:1]) was pumped onto the dried tissue over a period of about 5 s using a Nanoliter injector (World Precision Instruments, Berlin, Germany). Each preparation was air-dried and covered with pure water for a few seconds, which was removed by cellulose paper.
2.3.
MALDI-TOF mass spectrometry
MALDI-TOF analyses were performed on an ABI 4800 proteomics analyzer (Applied Biosystems, Framingham, MA). Due to the nature of the samples all acquisitions were taken in manual mode. Initially the instrument was operated in reflectron mode, in order to determine the parent masses. A laser intensity of 3800 was typically employed for ionizing the neuropeptides. For the tandem MS experiments (performed in gas on and gas off mode, respectively), the CID acceleration was 1 kV in all cases. The number of laser shots used to obtain a spectrum varied from 600 to 4000, depending on signal quality. The fragmentation patterns were used to manually determine the sequence of the peptides by using the Data ExplorerT software package.
3.
Results and discussion
Initially, synganglia of I. scapularis were dissected, the ganglionic sheath removed and these preparations used for MALDI-TOF mass spectrometry. The resulting mass spectra revealed only a few ion signals and the ion intensity was generally low. For that reason, synganglia were cut in smaller pieces of approx. 100 µm. These pieces were then directly transferred to a sample plate for MALDI-TOF mass spectrometry (see Fig. 1). Only mass spectra from such preparations yielded ion intensities that were sufficient for tandem mass spectrometry (Fig. 2A). In the mass range typical of shorter neuropeptides (600–3500 Da), numerous putative neuropeptides were detectable (Table 1). All prominent ions were fragmented by tandem mass spectrometry using the same samples that were analyzed for mass fingerprints before (see Fig. 3). Using the information from ESTs (see [1]), we could assign a large number of ion signals to predicted neuropeptides (see Fig. 2B,C), including RFamides (sulfakinins, short neuropeptide F), diuretic hormone (DH-35), corazonin, orcokinins, CAPA-periviscerokinin, SIFamides, tachykininrelated peptides (TKRP), and proctolin. In addition, the recently predicted allatostatin-C [11] and myoinhibitory peptide (MIP) [6]; were found to be expressed. Nearly all peptides which were observed in our mass spectra belong to neuropeptide families that are known to activate specific receptors in
Fig. 2 – Representative MALDI-TOF mass spectrum of a piece of tissue from the synganglion of I. scapularis. A: Mass range of 600–3500 Da. B: Detail of A, which illustrates the high number of ion signals in the mass range of 600–1800 Da. C: Detail of A which shows the ion signal of the diuretic hormone (DH-35). All prominent ion signals were analyzed by tandem mass spectrometry, successfully assigned neuropeptides are labelled. SK: sulfakinin; sNPF: short neuropeptide F; cor: corazonin; Orc: orcokinin; PK: pyrokinin; PVK: periviscerokinin; Proc: proctolin; TKRP: tachykinin-related peptide; MIP: myoinhibitory peptide.
insects (see [12]). The numerous additional neuropeptides that were predicted in Christie (2008) from the same precursors were not found [1]. Altogether, 20 neuropeptides from 12 neuropeptide families have been detected by this first comprehensive neuropeptidomic approach on ticks. More neuropeptides such as insect kinin-related peptides can be expected since the putative kinin receptor is already identified [13]. The prediction that the neuropeptidome of these arthropods is highly unique in comparison to other sequenced invertebrate genomes could not be confirmed. Indeed, the sequences of the tick neuropeptides show a close relationship with insect neuropeptides (e.g. Periplaneta americana, Drosophila melanogaster, Apis mellifera) although the evolutionary distance between ticks and insects is considerable. The class Insecta belongs phylogenetically to the subphylum Mandibulata and is closely related with Crustacea. The ticks belong to
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Table 1 – Summary of the neuropeptides that were detected in the CNS of I. scapularis by MALDI-TOF MS. Peptide name Corazonin Diuretic hormone-35 sNPF sNPF3-11 Sulfakinin-1 Sulfakinin-2 Orcokinin-2 Orcokinin-3 Orcokinin-PP proctolin Periviscerokinin Pyrokinin-1 Pyrokinin-2 Pyrokinin-3 SIFamide TKRP-1 (2x) TKRP-2 Allatostatin-C Myoinhibitory peptide Pyrokinin-4
Peptide sequence
[M + H]+
MS
MS/MS
EST hits
pQTFQYSRGWTNa AGGLLDFGLSRGASGAEAAKARLGLKLANDPYGPa GGRSPSLRLRFa SPSLRLRFa QDDDYGHMRFa SDDYGHMRFa WYGHGDFDEIDNVGWPGFT-OH NFDEIDRTGFEGFY-OH AANAAAAPAAREDA-OH RYLPT-OH pQGLIPFPRVa RSNNFTPRIa GSFVPRLa GSFTPRIa AYRKPPFNGSIFa AFHAMRa GSGFFGMRa SGWKQCSFNAVSCFa ASDWNRLSGMWa TPFTPRLa
1369.63 3312.78 1244.73 974.58 1282.50 1126.47 1623.67* 1709.74 1269.62 649.36 1008.59 1103.61 774.46 776.44 1395.75 731.38 857.41 1562.68 1321.61 830.54
x x x x x x x x x x x x x x x x x x x x
x x x x x x – x x x x x x x x x x – x x
EL516967 EW915260 EW855501 EW941557 EW941557 EW865036
EW933575
EW859993 EL516783
sNPF, short neuropeptide F; PP, precursor peptide; TKRP, tachykinin-related peptide. *[M + Na]+ and [M + K]+ adduct ions only.
the subphylum Chelicerata (together with e.g. spiders, scorpions, and mites) which separated from the Mandibulata nearly 500 million years ago [14] and it comes therefore as a surprise that the neuropeptides of these only distantly related arthropods are that similar. Certainly, the similarity of neuropeptide sequences within the large phylum Arthropoda (with more than 1 million species) will make it quite difficult to identify novel targets for tick and tick-borne disease control by developing specific peptide mimetics. An interesting phenomenon is the sequence of a peptide precursor that contains the CAPA-periviscerokinin (PVK). In insects, the capa-gene usually contains several PVKs and a single pyrokinin with the C-terminal WFGPRLa [15]. In
+
Fig. 3 – CID mass spectrum of the peptide at [M + H] : 649.4 Da under condition of high gas. The fragments were analyzed manually and the resulting sequence is given in the inset. The y-, a- and b-type ions, which verified the amino acid sequence of proctolin, are labelled.
addition, a second gene always encodes for other FXPRLamides (pyrokinins, pheromone biosynthesis activating neuropeptides [16]. It seems possible that the capa-gene and the pyrokinin gene of insects evolved from a common ancestor gene, at least the relationship of their specific receptors suggests so [17–19]. The sequence-related tick precursor contains a PVK typical of the capa-gene of insects and three pyrokinins typical of the fxprl-gene. It can thus be speculated, that this gene is an ancestor of the two insect genes and the differentiation in capa and fxprl-genes occurred after the separation of arthropods from the other taxa. However, by de
Fig. 4 – CID mass spectrum of the substance at [M + H]+: 830.5 Da. The y-, a- and b-type ions, which were used to identify the amino acid sequence of a pyrokinin (PK-4), are labelled. The deduced sequence of TPFTPRL-NH2 could not be assigned to any of the existing tick ESTs but shows a very close relationship to insect pyrokinins (see Table 2).
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Table 2 – Amino acid sequences of Ixodes pyrokinin-4 and sequence-related FXPRLamides from different insect taxa. Species Lepisma saccharina Periplaneta americana Nezara viridula Tribolium castaneum Drosophila melanogaster Apis mellifera Ixodes scapularis
Peptide sequence
References
SPPFAPRLa SPPFAPRLa SPPFAPRLa SPPFAPRLa SVPFKPRLa IYLPLFASPLa TPFTPRLa
Unpublished [20] [21] [19] [17] [22] This study
Acknowledgments The authors wish to thank Allison Strey for technical assistance (College Station, TX). This study was supported by a grant from Deutsche Forschungsgemeinschaft (Predel 595/6-4), and a grant from the USDA/DOD DWFP Initiative (#0500-32000-001-01R) (RJN).
REFERENCES novo sequencing (Fig. 4), we found a tick pyrokinin (TPFTPRLa) which is not encoded on the above-mentioned capa/pyrokinin gene. The sequence of this pyrokinin is very closely related to the most conserved pyrokinin of insects (see Table 2) and suggests the occurrence of a second fxprl-gene in ticks that has not yet been predicted. The peptidomics approach which was performed on I. scapularis, was subsequently repeated with another tick species, Amblyomma americanum. The resulting mass spectra verified a very close relationship of these ticks, and most neuropeptides were identical in both species (see Fig. 5). We found no trace of opioid peptides that were isolated as abundant peptides from synganglia of other tick species [7,8]. Possibly, these peptides were indeed contaminations from mammalian blood, and the intestine of ticks crosses the synganglion. The data presented here will provide a basic resource for studying tick biology. The differentiation between predicted and biochemically identified neuropeptides will help to avoid experiments with putative peptides with uncertain biological relevance.
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Fig. 5 – Comparison of MALDI-TOF mass spectra of preparations from synganglia of I. scapularis and A. americanum (mass range 700–1500 Da). Most neuropeptides are identical in both species; the sequences of the modified Amblyomma PK-1, and 2 are RSNTFTPRI-NH2 and STPVPRL-NH2.
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