Tachykinin-related peptide precursors in two cockroach species Molecular cloning and peptide expression in brain neurons and intestine Reinhard Predel1, Susanne Neupert1, Steffen Roth1, Christian Derst1 and Dick R Nassel2 ă Saxon Academy of Sciences, Research Group Jena, Germany Department of Zoology, Stockholm University, Sweden Keywords brain-gut peptides; insect neuropeptide; neurochemistry; mass spectrometry; Periplaneta americana; Leucophaea maderae Correspondence R Predel, Saxon Academy of Sciences, Research Group Jena, Erbertstraße 1, 07743 Jena, Germany Tel: +49 3641 949191 Fax: +49 3641 949192 E-mail: B6PRRE@pan.zoo.uni-jena.de (Received March 2005, revised 22 April 2005, accepted May 2005) doi:10.1111/j.1742-4658.2005.04752.x Tachykinins and tachykinin-related peptides (TKRPs) play major roles in signaling in the nervous system and intestine of both invertebrates and vertebrates Here we have identified cDNAs encoding precursors of multiple TKRPs from the cockroaches Leucophaea maderae and Periplaneta americana All nine LemTKRPs that had been chemically isolated in earlier experiments could be identified on the precursor of L maderae Four previously unidentified LemTKRPs were found in addition on the precursor The P americana cDNA displayed an open reading frame very similar to that of L maderae with 13 different TKRPs MALDI-TOF mass spectra from tissues of both species confirms the presence of all the TKRPs encoded on the precursor plus two additional peptides that are cleavage products of the N-terminally extended TKRPs A tissue-specific distribution of TKRPs was observed in earlier experiments at isolation from brain and midgut of L maderae Our data not suggest a differential gene expression but a different efficacy in processing of LemTKRP-2 and Lem ⁄ PeaTKRP-3 in the brain and intestine, respectively This results in a gut-specific accumulation of these extended peptides, whereas in the brain their cleavage products, LemTKRP-1 and LemTKRP-311)19, are most abundant Mass spectrometric analysis demonstrated the occurrence of the different TKRPs in single glomeruli of the tritocerebrum and in cells of the optical lobe Tachykinins constitute a family of multifunctional neuropeptides whose signaling mechanisms seem to be partially conserved through evolution [1–5] Although the tachykinin peptides display only limited sequence identities when comparing invertebrates and mammals, their G-protein-coupled receptors (GPCRs) display more striking similarities, suggesting ancestral relationships [4,5] The three principal mammalian tachykinins, substance P, neurokinin A and neurokinin B, are processed from two precursors, preprotachykinin A and B and they act with preferential affinities on three different GPCRs, NK1–NK3 [5] More recently, additional tachykinins, the hemokinins, were identified on a third precursor encoding gene, preprotachykinin C, expressed in hematopoietic cells of mouse, rat and humans [6,7] In invertebrates the tachykinins exist in two major forms: (a) the tachykinin-related peptides (TKRPs; previously termed TRPs) that differ from the mammalian tachykinins by having a C-terminus FXGXRamide (X ¼ variable residues), rather than FXGLMamide and (b) invertebrate tachykinins (Inv-TKs) with an Abbreviations ESI-Q-TOF MS, electrospray ionization quadrupole time-of-flight mass spectrometry; GPCR, G-protein-coupled receptors; TKRP, tachykininrelated peptide FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS 3365 Tachykinin-related peptides in cockroaches FXGLMamide C-terminus [2,4] As there is no evidence that the insect and molluscan Inv-TKs display biological activity in the native organism, it is likely that the TKRPs are the principal endogeneous tachykinins in invertebrates The TKRPs are known to exist in multiple forms encoded by a single gene in each species studied so far [2,4] Thus there are six TKRPs encoded on the Drosophila melanogaster gene dtk [8], seven in the honey bee Apis mellifera [9], three in the mosquito Anopheles gambiae [10], seven in the echiuroid worm Urechis unicinctus [11,12], and seven identical copies of a single form in the crayfish and spiny lobster [13] The largest number of TKRP isoforms in a single species was isolated biochemically from the cockroach Leucophaea maderae In this species nine different TKRPs (LemTKRP-1–9) were identified [14,15] Immunocytochemistry revealed the distribution of LemTKRP-like immunoreactivity in numerous neurons of the central nervous system and peripheral ganglia, as well as in the intestine and in some neurons innervating skeletal muscles [16] With antisera specific to several LemTKRPs (1,2,7) it could be demonstrated that these are colocalized in the same neurons of the brain [17] Earlier data also indicated that some isoforms are expressed only in the brain (LemTKRP6,7,8,9) or intestine (LemTKRP-3,4), and some in both tissues (LemTKRP-1,2,5) [14,15] To be able to solve the question of cell- or tissue-specific expression of LemTKRPs it is critical to identify the gene(s) encoding their precursor(s) In the present paper, we have cloned the genes encoding the TKRPs of L maderae and the American cockroach Periplaneta americana MALDI-TOF mass spectra from tissues of both species suggest a different efficacy in processing of two N-terminally extended TKRPs in the brain and intestine, respectively This could result in the gut-specific accumulation of such peptides, whereas in the brain cleavage products of these TKRPs are abundant Apart from these, all TKRPs that can be predicted from the precursors of the two species were found in the brain as well as in the midgut In total, 14 different TKRPs can be generated in L maderae and 15 in P americana Mass spectrometric analysis demonstrated the occurrence of the different TKRPs in glomeruli of the tritocerebrum and in cells of the optical lobe Results Cloning of cockroach preprotachykinin cDNAs Using degenerated PCRs and RACE, we obtained full length preprotachykinin cDNA sequences for L maderae and P americana The cDNAs of 1512 bp 3366 R Predel et al (lem, GenBank accession number AY766011) and 1200 bp (pea, GenBank accession number AY766012) code for open reading frames of 360 amino acids (L maderae) and 366 amino acids (P americana) Sequence analysis identified a signal peptide (26aa) and 13 tachykinin-related peptides (TKRPs) in both sequences As biochemical data showed alternative processing of the first TKRP sequence (giving rise to LemTKRP-1 and LemTKRP-2) more TKRPs may be processed out of the precursor molecule The sequences of the identified TKRPs are highly conserved between the two cockroach species, 95.1% of the 144 amino acids encoding for mature TKRPs are identical in L maderae and P americana, only six amino acid substitutions and one deletion were found (Fig 1) The signal peptide sequence (69.2% identity) and the spacer sequences between the TKRPs (64.8% identity) are much less conserved (Fig 1) When comparing the individual TKRP sequences, 10 of 13 peptides are nona- or decapeptides; only the unprocessed first and the last encoded peptide are significantly longer (TKRP-2, 18 amino acids and TKRP-3, 19 amino acids) In addition, a novel peptide (TKRP-11) consisting of only seven amino acids was found Nearly all TKRP sequences are followed by typical amidation ⁄ processing signals (GKK motif) within the precursor, only LemTKRP-4 was followed by the GKR motif Mass spectrometric screening of TKRPs in brain neurons and tritocerebral glomeruli of P americana and L maderae Immunocytochemical results have shown a number of TKRP immmunopositive cells in the brain of L maderae [16] Mainly due to good accessibility, immunoreactive glomeruli of the tritocerebrum and neurons in the vicinity (inner side) of the accessory medulla were chosen for mass spectrometric analysis The tritocerebral neuropil receives sensory input from the labrum via the labral nerves [18] Cells and glomeruli were individually dissected for both species and prepared for MALDI-TOF MS Resulting mass spectra from tritocerebral glomeruli suggested that these glomeruli contain the majority of the TKRPs encoded on the precursors (Fig 2) The orthologs LemTKRP-3 ⁄ PeaTKRP-3 could not be detected in any preparation where an otherwise good signal-to-noise ratio was obtained However, a cleavage product of TKRP-3 (LemTKRP-311)19) was found in both species From the other extended TKRP (LemTKRP-2) very weak ion signals could be detected only in a few preparations from L maderae (not FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS R Predel et al Tachykinin-related peptides in cockroaches Fig Amino acid sequences of the P americana (Pea) and L maderae (Lem) TKRP precursors Identified peptides are labeled in gray; designations not follow (for historical reasons) the position in the precursors, thus numbering of the TKRPs is given below the amino acids As seen in the consensus sequence in the middle, only six amino acid substitutions and one deletion were found in the peptide-encoding regions shown) Glomeruli of both species, however, contained a cleavage product of TKRP-2 (LemTKRP-29)17) which is identical to LemTKRP-1 None of the numerous known cockroach peptides was observed in mass spectra of tritocerebral glomeruli from the two species, but a number of unknown substances were represented (Fig 2) In a second step, glomeruli (50–60 lm) of the tritocerebrum were individually dissected (Fig 3) In these experiments, it could be confirmed that each of the TKRP containing glomeruli indeed contained all the peptides found in the complete cluster of tritocerebral glomeruli (Fig 4) Mass spectra of TKRP-producing cells in the optic lobe of both cockroach species revealed the presence of all TKRPs found in the tritocerebral glomeruli (Fig 5) This suggests that the peptide pattern of TKRPs in the brain is not site-specific Again, the two extended TKRPs (TKRP-2 and -3) were not detectable Mass spectra of these cells (15–20 lm) showed a much lower signal intensity than those of the tritocerebral glomeruli The adjoining unknown substances typical of the tritocerebral TKRP-glomeruli were also found in mass spectra from preparations of optic lobe cell bodies To confirm the identity of the mass signals FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS of the TKRPs proposed from the precursor, an extract of the tissue from 50 tritocerebral neuropil areas of P americana was prepared for ESI-Q-TOF MS The doubly charged ion species of 534.3 ([M + H]+ of 1067.6) was chosen for tandem fragmentation, as it was suspected to contain two TKRPs with identical masses (LemTKRP-7 and -12) whose expression could not be verified with MALDI-MS Although the full scan of the solution after rinsing the purification capillary with 20% acetonitrile ⁄ 5% formic acid revealed only a very weak signal at 534.3 (with the other TKRPs mostly below the threshold), the CID spectrum clearly confirmed that both peptides were present in the sample (Fig 6) The peptide sequences, designations and masses of all TKRPs from both cockroach species are listed in Table Screening for TKRPs in the midgut of P americana A midgut-specific expression of TKRPs was found for L maderae in earlier experiments [15] Our analysis of TKRP-expressing neurons of the brain indicates that only the long TKRPs (TKRP-2 and -3) are candidates for midgut-specific expression As a direct mass 3367 Tachykinin-related peptides in cockroaches R Predel et al Fig MALDI-TOF mass spectra typical of tritocerebral TKRP-containing glomeruli TKRPs are numbered (A) L maderae, (B) P americana spectrometric screening of peptides from gut tissues failed, we analyzed fractions obtained after HPLCseparation of an extract from 20 midguts of P americana MALDI-TOF mass spectra revealed the occurrence of all TKRPs which were detected in the brain neurons also in the midgut This includes the aforementioned cleavage products of TKRP-2 and -3 In addition, distinct mass signals typical of unprocessed TKRP-2 and TKRP-3 were observed All TKRPs 3368 typical of P americana (PeaTKRP-3,4,6,9,10,14) were subsequently chosen for tandem fragmentation which confirmed the predicted sequences (not shown) Discussion Studies using cockroaches greatly contributed to our knowledge about neuropeptides of invertebrates It was with L maderae that the identification of many FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS R Predel et al Fig Immunofluorescence staining in the deuto- (DC) and tritocerebrum (TC) of P americana by means of an antiserum against Lom-TKRP (whole mount preparation) Immunoreactivity is mainly detectable in glomeruli of the antennal lobe (AL) and the tritocerebral neuropil (arrow) which receives input from the labral nerves Scale bar: 500 lm The inset shows an isolated tritocerebral glomerulus, which was subsequently analyzed by MALDI-TOF MS (Fig 4), immediately before transfer to the sample plate novel neuropeptide families started in the 1980s [19] These findings provided the basis for the subsequent detection of neuropeptides in other insect species, among them the American cockroach, locusts and also D melanogaster [20,21,22] In this study, we have cloned tachykinin-related peptide encoding cDNAs from P americana and L maderae Today, with the present identification of the TKRPs from P americana, no other insect species is known to express a larger number of identified neuropeptides (more than 80 peptides [20,23]) in the CNS Both cockroach TKRP precursors contain 13 copies of related TKRPs and no other predicted peptides with amidation signals The L maderae precursor contains two copies of LemTKRP-9 and single copies of the other 11 peptides, whereas in P americana there are 13 different TKRPs We also showed that two additional peptides (LemTKRP-1 and LemTKRP-311)19) can be cleaved from these progenitor sequences in both species, producing 15 different TKRPs in P americana and 14 in L madFEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS Tachykinin-related peptides in cockroaches erae This number of TKRPs is large compared to that in the precursors in the worm U unicinctus, crayfish, other insects and mammalians and seems to be in line with data from other known cockroach neuropeptide precursors For instance the allatostatin-A type precursors of different cockroaches display a higher number of allatostatin forms than those in D melanogaster and A gambiae [10,24], and the recently identified precursor for FMRFamide-related peptides of P americana contains the largest number of neuropeptides known to be encoded on a single precursor in any insect [20] Although P americana and L maderae belong to different suborders of the order Blattodea, their TKRP precursors display striking similarities in their peptide encoding regions (Fig 1) Most TKRPs are identical, and others display minor amino acid substitutions Two N-terminally extended peptides (TKRP-2 and -3) are present in both species and the other peptides are nona- or decapeptides, except LemTKRP-11 which is a heptapeptide It is also noteworthy that all the LemTKRPs and their P americana orthologs, except LemTKRP-3 and 11, have a proline in the second position (XPX-) of their N-terminus This proline provides some resistance to nonspecialized amino peptidases, but also renders the peptides sensitive to proline-specific dipeptidyl peptidase (DPP IV) attack [25,26] A comparison with the open reading frames of other invertebrate TKRP encoding genes reveals that the largest similarities are in the actual peptide progenitor sequences, whereas signal peptides and spacing sequences between peptides are much more divergent Between the two cockroach genes the similarities are also much less distinct in the nonpeptide coding regions (64.8% similarities in spacer regions, 69.8% similarities in signal peptide) Compared to other cloned TKRP precursors there are some major differences in nonpeptide coding sequences For instance in the worm U unicinctus and the mosquito A gambiae, the TKRP sequences are separated only by the dibasic amino acids and amidation signals [10,12]; whereas in the cockroaches, honeybee [9], crayfish, spiny lobster [13] and fruit fly [8], there are sequences of varying length between the TKRPs Only in the D melanogaster TKRP precursor, Dtk, other putative amidated peptides (unrelated to TKRPs) could be predicted It is, however, not likely that these additional putative peptides are cleaved from the Drosophila precursor [27] The most N-terminally located peptide LemTKRP-2 does not have dibasic cleavage sites N-terminally Thus this peptide has to be cleaved directly from the signal peptide (probably by a signal peptidase) A similar 3369 Tachykinin-related peptides in cockroaches R Predel et al Fig MALDI-TOF mass spectrum typical of a single tritocerebral TKRP-containing glomerulus of L maderae; TKRPs are numbered Both ion species and relative abundances are very similar to spectra from preparations of complete TKRP-containing neurophils (Fig 2A) Fig MALDI-TOF mass spectrum typical of TKRP-containing cells in the optic lobe of P americana TKRPs are numbered Ion intensities from these preparations are much lower than those found in spectra from tritocerebral glomeruli The relative abundance of the different TKRPs, however, is comparable with the situation in tritocerebral glomeruli unusual location adjacent to the signal peptide was for instance seen for the neuropeptide proctolin in D melanogaster [28] Another feature of the LemTKRP-2 sequence (17-mer) is that it contains a dibasic cleavage site at which the peptide can be cleaved to obtain the 9mer LemTKRP-1 This cleavage does occur, as we could demonstrate both LemTKRP-1 and LemTKRP2 in the P americana midgut in this investigation and both peptides were chemically isolated from the brain and intestine of L maderae [14,15] The other N-ter3370 minally extended peptides LemTKRP-3 and PeaTKRP-3 (19mers) also contain a dibasic cleavage signal (Lys-Lys), and in the brain only the truncated version was observed in both species, whereas the 19-mer was identified in the L maderae [15] and P americana midgut Hence, with the extended TKRP-2 and -3 as exceptions, all of the predicted cockroach TKRPs could be reproducibly detected by MALDI-TOF MS in tritocerebral glomeruli and cells near the accessory FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS R Predel et al Tachykinin-related peptides in cockroaches Fig CID (collision-induced dissociation) spectrum (600–1000 atomic mass units) of TKRPs at [M + 2H]+ of 534.3 ([M + H]+:1067.6; Fig 3B) from a methanolic extract of 50 tritocerebral glomeruli using a nanospray source Before this experiment, the sample was purified on a capillary filled with Luna C18 material and the spectrum was taken after rinsing the capillary with 20% (v ⁄ v) acetonitrile ⁄ 5% (v ⁄ v) formic acid Only the y-type fragment ions in the higher mass range are given The fragments clearly confirmed the presence of TKRP-7 (white labeling on dark background) and 12 (dark labeling on white background) in the sample that could otherwise not be separated due to mass identity medulla of the optic lobe The unequivocal detection of TKRPs in single neurons and even glomeruli of the brain demonstrates the power of modern MS technology when combined with proper identification and sample preparation Mass spectra also provide the evi- dence that interneurons may contain amounts of neuropeptides comparable with those of neuroendocrine cells which produce peptide hormones such as FMRF amide-related peptides-producing cells in the thoracic ganglia [20] Earlier studies on L maderae dealt with the problem of brain-gut peptides and suggested a differential distribution of some TKRPs (see introduction) As shown in this study, no brain-specific TKRP-gene products exist However, the N-terminally extended TKRPs could not (Lem ⁄ PeaTKRP-3), or only with very low signal intensity (LemTKRP-2), be identified in the brain by mass spectrometry Thus, the previously documented absence in the brain of LemTKRP-3 [14,17] could be confirmed in L maderae and was also true for P americana The trace amounts of LemTKRP-2 in the brain of L maderae, however, indicate that the differential distribution of the extended TKRPs and their truncated forms may be attributed mainly to a differential efficacy in further cleavage of TKRP-2 and -3 Thus, the unprocessed TKRP-2 and -3 are probably present in the brain, but in a much lower concentration than in the midgut and require some biochemical enrichment to be detectable This is the likely explanation why LemTKRP-2 could be chemically isolated from both brain and intestine in L maderae [14,15] The finding that N-terminally extended TKRPs are predominantly expressed only in the gut and not in the brain are in line with findings of enrichment of Table Peptide sequences, designations and masses of all TKRPs from L maderae and P americana To fit with the P americana orthologs, LemTKRP-9 with two identical copies on the precursor occurs twice The truncated versions of TKRP-2 and -3 are included in the table; for historical reasons the cleavage product of TKRP-2 (TKRP-29)17) retains its own designation, namely LemTKRP-1 Non-identical amino acids are indicated in bold Leucophaea maderae Periplaneta americana Peptides Peptide sequence [M + H]+, m ⁄ z Peptides Peptide sequence [M + H]+, m ⁄ z LemTKRP-1 LemTKRP-2 LemTKRP-3 LemTKRP-311)19 LemTKRP-4 LemTKRP-5 LemTKRP-6 LemTKRP-7 LemTKRP-8 LemTKRP-9 LemTKRP-10 LemTKRP-11 LemTKRP-12 LemTKRP-13 LemTKRP-9 APSGFLGVRa APEESPKRAPSGFLGVRa NGERAPGSKKAPSGFLGTRa APSGFLGTRa APSGFMGMRa APAMGFQGVRa APAAGFFGMRa VPASGFFGMRa GPSMGFHGMRa APSMGFQGMRa GPNMGFMGMRa MGFMGMRa GPSVGFFAMRa APSAGFMGMRa APSMGFQGMRa 902.52 1796.98 1929.04 904.50 952.45 1032.54 1023.52 1067.55 1075.49 1080.51 1096.48 828.36 1067.55 1023.49 1080.51 LemTKRP-1 LemTKRP-2 PeaTKRP-3 LemTKRP-311)19 PeaTKRP-4 LemTKRP-5 PeaTKRP-6 LemTKRP-7 LemTKRP-8 PeaTKRP-9 PeaTKRP-10 LemTKRP-11 LemTKRP-12 LemTKRP-13 PeaTKRP-14 APSGFLGVRa APEESPKRAPSGFLGVRa NGERAPASKKAPSGFLGTRa APSGFLGTRa APGSGFMGMRa APAMGFQGVRa APASGFFGMRa VPASGFFGMRa GPSMGFHGMRa APSLGFQGMRa APNMGFMGMRa MGFMGMRa GPSVGFFAMRa APSAGFMGMRa APSAGFHGMRa 902.52 1796.98 1943.06 904.50 1009.47 1032.54 1039.51 1067.54 1075.49 1062.55 1110.50 828.36 1067.55 1023.49 1029.50 FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS 3371 Tachykinin-related peptides in cockroaches N-terminally extended tachykinins, neuropeptide c and neuropeptide K, in the mammalian intestine [29–31] Another extended TKRP was isolated from the locust intestine [32] In mammals, the tissue-specific expression of tachykinins is caused by alternative splicing of the PPT-A gene and the existence of two further PPT genes, but probably also by differential post-translational processing [5] Previous work has indicated that the different LemTKRPs and the D melanogaster and Locusta migratoria TKRPs display very small differences in their biological activity in different bioassays [2,4,8,22,33] As the TKRPs are encoded on a single precursor and expressed in the same tissue (or even cells), it is possible that several of the TKRPforms are physiologically redundant This, however, needs to be carefully investigated One reason for this is that although only one putative TKRP receptor is known so far from L maderae [34], an additional GPCR of the tachykinin type has been identified in some other insects [35–38] In summary, we have found that cockroach genes for TKRP precursors are the ones encoding the largest number of copies of different TKRPs of all invertebrates studied so far Most of the 14–15 peptides predicted on each of the precursors could be identified in the brain and intestine of the two cockroach species by mass spectrometry A possible case of tissue-specific accumulation of the N-terminally extended peptides encoded on the genes could be confirmed by mass spectrometry Experimental procedures R Predel et al part of the preprotachykinin precursors from L maderae and P americana cDNA: forward primer: 5¢-gcnccng cnatgggnttycarggngt-3¢ encoding for APAMGFQGV (part of TKRP5); reverse primer: 5¢-ggngcyttyttnswnccnggngcnck ytcnccrtt-3¢ encoding for NGERAPASKKA (part of TKRP3) The partial preprotachykinin cDNA sequences obtained were used to design primers for 3¢ and 5¢ nested rapid amplification of cDNA ends (RACE): lemRACE-F1: 5¢-TCGCTGTTGCAGTACCTGGACTCC-3¢; lemRACEB1: 5¢-GTCTACCAAGTCTCGAAGAAAGTCCTGCTG3¢; peaRACE-F1: 5¢-GATGGAGGGCGCGGAGGAT-3¢; peaRACE-B1: 5¢-CTTGCCCCTCATGCCATGGAAC-3¢ For both RACE reactions we used Advantage Taq Mixture (Clontech) and a P americana RACE library constructed previously [39] A L maderae RACE library was prepared from several tissues (brain, ganglia, malpighian tubules, muscles and intestine) using Trizol Reagent (Invitrogen, Karlsruhe, Germany) for RNA preparation, Oligotex (Qiagen, Hilden, Germany) for polyA + RNA preparation and the Marathon cDNA Amplification Kit (Clontech, Heidelberg, Germany) for reverse transcription, second strand synthesis and adapter ligation RACE products were cloned into pGEM-T vector (Promega GmbH, Mannheim, Germany) for sequencing Sample preparation for mass spectrometry After dissection of the brain, the ganglionic sheath was partially removed, glomeruli or somata of neurosecretory cells separated and transferred with the help of a glass capillary to a stainless steel sample plate for MALDI-TOF MS or into a chilled solution of lL methanol ⁄ water ⁄ trifluoroacetic acid (90 : : 1, v ⁄ v ⁄ v) for electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) Insects Cockroaches, L maderae and P americana, were raised under a 12 h light, 12 h dark photoperiod at a constant temperature of 28 °C They were fed food pellets for rats and had free access to water Adult cockroaches of both sexes were used for experiments Cloning of the L maderae and P americana TKRP precursor cDNA A combined degenerated PCR and RACE strategy was used to clone full length sequences of cockroach preprotachykinin cDNAs as described recently for preproFMRFamide cDNA [20] Based on biochemically identified L maderae TKRPs [14,15] several degenerated PCR primers were designed PCR reactions were performed with Advantage Taq Mixture (Clontech, Palo Alto, CA, USA) at low stringency Among the primer combinations tested one pair of primers amplified approximately 790 bp fragments encoding 3372 MALDI-TOF MS Neurons ⁄ glomeruli were dried on the sample plate and subsequently rinsed with water to reduce salt contamination Matrix solution (a-cyano-4-hydroxycinnamic acid dissolved in methanol–water) was pumped onto the dried preparations over a period of approximately s using a nanoliter injector (World Precision Instruments, Berlin, Germany) Each preparation was allowed to dry and then covered with pure water for a few seconds; the water was then removed by cellulose paper At least five preparations each were prepared for mass spectrometric analysis ESI-Q-TOF MS Following the dissection procedure, 50 lL 0.1% (v ⁄ v) trifluoroacetic acid were added to the lL methanol ⁄ water ⁄ trifluoroacetic acid The extract was sonicated, centrifuged FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS R Predel et al and the methanol evaporated from the supernatant The resulting aqueous supernatant was then loaded onto an activated and equilibrated home-made micro column (purification capillary for electrospray mass spectrometry) MALDI-TOF MS MALDI-TOF mass spectra were acquired in positive ion mode on a Voyager Pro DE biospectrometry workstation (Applied Biosystems, Framingham, MA, USA) equipped with a pulsed nitrogen laser emitting at 337 nm Samples were analyzed in reflectron mode using a delayed extraction time of 150 ns, 75% grid voltage, 0.002–0.02% guide wire voltage, and an accelerating voltage of 20 kV Laser strength was adjusted to provide the optimal signal-to-noise ratio An external mass spectrum calibration was first performed using synthetic cockroach peptides (Pea-pyrokinin2 ⁄ 5; SPPFAPRLa ⁄ GGGGSGETSGMWFGPRLa) ESI-Q-TOF MS Nanoelectrospray mass spectra were acquired in the positive-ion mode using the API Qstar Pulsar (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) fitted with a Protana (Odense, Denmark) nanoelectrospray source Typically 1050–1150 V was applied as an ion spray voltage Samples were purified using a homemade spin column Approximately mg of Luna C18 material (10 lm; Phenomenex, Aschaffenburg, Germany) was loaded into a 2-cm capillary column with a needle tip Liquids are passed through the column by securing the capillary column to a purification needle holder (Proxeon Biosystems A ⁄ S, Odense, Denmark) and centrifugation After the column was activated with 50% acetonitrile ⁄ 0.1% TFA and equilibrated in 0.1% TFA, the samples were loaded and rinsed with 5% formic acid Peptides were eluted from the column with solutions of 10, 20, and 30% acetonitrile (5% formic acid) and collected into a metal-coated nanoelectrospray capillary The purified samples were then loaded onto the source and analyzed After determining the m ⁄ z of the peptides in MS mode, a collision energy (typically 15–40 V) was applied The m ⁄ z of interest was isolated and fragmented with the instrument in ‘enhance all’ mode MS ⁄ MS data were acquired over and manually analyzed HPLC Midguts of adult P americana were dissected in insect saline and shortly rinsed with distilled water before being transferred to 200 lL methanol–water–trifluoroacetic acid (90 : : 1, v ⁄ v ⁄ v, on ice) Following sonication and centrifugation, the collected supernatant was evaporated to dryness and resuspended in 500 lL 0.1% (v ⁄ v) trifluoroacetic acid This solution was applied to an activated and equilibrated FEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS Tachykinin-related peptides in cockroaches SEP-PAK C-18 cartridge (Waters, Milford, MA, USA), peptides were eluted with 40% (v ⁄ v) acetonitrile containing 0.1% (v ⁄ v) trifluoroacetic acid Peptide separation was performed on a Shimadzu HPLC system (Shimadzu, Duisburg, Germany) equipped with a diode-array detector and using a ˚ Luna RP-C18 column (150 · 4.6 mm, 100 A, lm, Phenomenex, Torrance, CA, USA) with a linear AB gradient of 10–80% B over 35 (flow rate: mLỈmin)1) Solvent A was 0.11% (v ⁄ v) trifluoroacetic acid in water, solvent B 60% (v ⁄ v) acetonitrile containing 0.1% (v ⁄ v) trifluoroacetic acid Fractions were collected manually and subsequently analyzed on a MALDI-TOF mass spectrometer Immunocytochemistry Dissected cockroach brains were fixed overnight at °C with 4% (v ⁄ v) formaldehyde in phosphate-buffered saline (NaCl ⁄ Pi), pH 7.2 Subsequently, preparations were washed in NaCl ⁄ Pi)4% (v ⁄ v) Triton X-100 and NaCl ⁄ Pi)1% (v ⁄ v) Triton X-100 for 24 h, respectively The preparations were then incubated for days at °C in anti-LomTKRP serum (1 : 1000, diluted with NaCl ⁄ Pi 1% (v ⁄ v) Triton X-100 containing 0.25% (w ⁄ v) bovine serum albumin and normal goat serum) Following overnight washing in 0.1 molỈL)1 Tris ⁄ HCl, 3% (w ⁄ v) NaCl, 1% (v ⁄ v) Triton X-100 (pH 7.6), the fluorochrome-labeled secondary Cy3 antibodies were used directly as a mixture in NaCl ⁄ Pi–bovine serum albumin (2.5 mgỈmL)1) at a concentration of : 3000 for days Finally, the preparations were washed again overnight in 0.1 molỈL)1 Tris-HCl, 3% (w ⁄ v) NaCl, 1% (v ⁄ v) Triton X-100 (pH 7.6) and transferred in glycerin For visualization, tissues were dehydrated in ethanol, cleared in methyl salicylate and mounted in entellan (Euromex Microscopes, Arnhem, the Netherlands) Immunostainings were examined with a confocal laser scanning microscope (Zeiss LSM 510 Meta system; Jena, Germany), equipped with a HeliumNeon1 laser (wavelength 543 nm) Serial optical sections were assembled into combined images Images were exported and processed with Adobe photoshop 7.0 software Peptide terminology and acronyms The old designation of tachykinin-related peptide (TRP) was changed here for TKRP as suggested in [4] This was done to avoid confusion with, for example, the abbreviation for the transient receptor potential cationic channel (TRP) and tryptophan (Trp) Thus the old acronyms such as LemTRP-1, were changed to LemTKRP-1 The designations of the P americana TKRPs were made such that peptides identical to L maderae peptides are called LemTKRPs, others are PeaTKRPs The numbering of peptides follows that for already identified peptides [14,15], and not their sequence order on the precursors Thus novel peptides 3373 Tachykinin-related peptides in cockroaches are given numbers TKRP-10–14 Unique PeaTKRPs are given numbers that are the same as their LemTKRP orthologs on the precursor Note that the nonamidated pepide designated LemTRP-10 in an earlier paper [14] is not likely to be a true TKRP and is disregarded here (thus the LemTKRP-10 identified here is a totally new peptide) Acknowledgements We 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electrophysiology Eur J Neurosci 17, 1197–1212 3375 ... from both brain and intestine in L maderae [14,15] The finding that N-terminally extended TKRPs are predominantly expressed only in the gut and not in the brain are in line with findings of enrichment... 3365–3375 ª 2005 FEBS 3371 Tachykinin-related peptides in cockroaches N-terminally extended tachykinins, neuropeptide c and neuropeptide K, in the mammalian intestine [29–31] Another extended... tachykinin and insect kinin ⁄ Tyr-W-MIF neuropeptide family pairs Ann NY Acad Sci 897, 388–400 Nassel DR (1999) Tachykinin-related peptides in inveră tebrates: a review Peptides 20, 141158 Vanden