MALDI-TOF mass spectra from tissues of both species confirms the presence of all the TKRPs enco-ded on the precursor plus two additional peptides that are cleavage prod-ucts of the N-term
Trang 1Molecular cloning and peptide expression in brain neurons and
intestine
Reinhard Predel1, Susanne Neupert1, Steffen Roth1, Christian Derst1and Dick R Na¨ssel2
1 Saxon Academy of Sciences, Research Group Jena, Germany
2 Department of Zoology, Stockholm University, Sweden
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
relation-ships [4,5] The three principal mammalian
tachyki-nins, 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, addi-tional 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; pre-viously termed TRPs) that differ from the mammalian tachykinins by having a C-terminus FXGXRamide (X¼ variable residues), rather than FXGLMamide
and (b) invertebrate tachykinins (Inv-TKs) with an
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 1 March 2005, revised 22 April
2005, accepted 6 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 ver-tebrates Here we have identified cDNAs encoding precursors of multiple TKRPs from the cockroaches Leucophaea maderae and Periplaneta ameri-cana All nine LemTKRPs that had been chemically isolated in earlier experiments could be identified on the precursor of L maderae Four previ-ously 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 enco-ded on the precursor plus two additional peptides that are cleavage prod-ucts 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 do 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
Abbreviations
ESI-Q-TOF MS, electrospray ionization quadrupole time-of-flight mass spectrometry; GPCR, G-protein-coupled receptors; TKRP, tachykinin-related peptide.
Trang 2FXGLMamide 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
mul-tiple forms encoded by a single gene in each species
stud-ied 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
inner-vating 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
iso-forms are expressed only in the brain
(LemTKRP-6,7,8,9) 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)
enco-ding 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
spe-cies suggest a different efficacy in processing of two
N-terminally extended TKRPs in the brain and
intes-tine, 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
occur-rence of the different TKRPs in glomeruli of the
trito-cerebrum 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
(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 sub-stitutions 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) consist-ing of only seven amino acids was found Nearly all TKRP sequences are followed by typical amida-tion⁄ processing signals (GKK motif) within the precur-sor, 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 mad-erae [16] Mainly due to good accessibility, immuno-reactive glomeruli of the tritocerebrum and neurons in the vicinity (inner side) of the accessory medulla were chosen for mass spectrometric analysis The tritocere-bral neuropil receives sensory input from the labrum via the labral nerves [18] Cells and glomeruli were indi-vidually 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 clea-vage 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
Trang 3shown) Glomeruli of both species, however, contained
a cleavage product of TKRP-2 (LemTKRP-29)17)
which is identical to LemTKRP-1 None of the
numer-ous 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
trito-cerebrum 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
detect-able Mass spectra of these cells (15–20 lm) showed a
much lower signal intensity than those of the
tritocere-bral glomeruli The adjoining unknown substances
typ-ical 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
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 capil-lary 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 spec-trum clearly confirmed that both peptides were present
in the sample (Fig 6) The peptide sequences, designa-tions and masses of all TKRPs from both cockroach species are listed in Table 1
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 candi-dates for midgut-specific expression As a direct mass
Fig 1 Amino acid sequences of the P americana (Pea) and L maderae (Lem) TKRP precursors Identified peptides are labeled in gray; des-ignations do 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.
Trang 4spectrometric screening of peptides from gut tissues
failed, we analyzed fractions obtained after
HPLC-separation of an extract from 20 midguts of P
ameri-cana 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
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
Fig 2 MALDI-TOF mass spectra typical
of tritocerebral TKRP-containing glomeruli TKRPs are numbered (A) L maderae, (B) P americana.
Trang 5novel 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
ameri-cana, no other insect species is known to express a
lar-ger 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
Lem-TKRP-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
(Lem-TKRP-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
mad-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 precur-sors of different cockroaches display a higher number
of allatostatin forms than those in D melanogaster and A gambiae [10,24], and the recently identified pre-cursor for FMRFamide-related peptides of P ameri-cana 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 TKRPs and their P americana orthologs, except Lem-TKRP-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-speci-fic 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 differ-ences in nonpeptide coding sequdiffer-ences 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 melano-gaster 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
Fig 3 Immunofluorescence staining in the deuto- (DC) and
tritocere-brum (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),
imme-diately before transfer to the sample plate.
Trang 6unusual location adjacent to the signal peptide was for
instance seen for the neuropeptide proctolin in D
mel-anogaster [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 1 and
LemTKRP-2 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-ter-minally extended peptides LemTKRP-3 and Pea-TKRP-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 americanamidgut
Hence, with the extended TKRP-2 and -3 as excep-tions, all of the predicted cockroach TKRPs could
be reproducibly detected by MALDI-TOF MS in tritocerebral glomeruli and cells near the accessory
Fig 4 MALDI-TOF mass spectrum typical
of a single tritocerebral TKRP-containing glomerulus of L maderae; TKRPs are num-bered Both ion species and relative abundances are very similar to spectra from preparations of complete TKRP-containing neurophils (Fig 2A).
Fig 5 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.
Trang 7medulla of the optic lobe The unequivocal detection
of TKRPs in single neurons and even glomeruli of the
brain demonstrates the power of modern MS
technol-ogy when combined with proper identification and
sample preparation Mass spectra also provide the
evi-dence that interneurons may contain amounts of neu-ropeptides 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 dis-tribution 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 Lem-TKRP-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
Fig 6 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
tritocere-bral 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
other-wise not be separated due to mass identity.
Table 1 Peptide sequences, designations and masses of all TKRPs from L maderae and P americana To fit with the P americana ortho-logs, 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 APSGFLGVRa 902.52 LemTKRP-1 APSGFLGVRa 902.52 LemTKRP-2 APEESPKRAPSGFLGVRa 1796.98 LemTKRP-2 APEESPKRAPSGFLGVRa 1796.98 LemTKRP-3 NGERAPGSKKAPSGFLGTRa 1929.04 PeaTKRP-3 NGERAPASKKAPSGFLGTRa 1943.06 LemTKRP-311)19 APSGFLGTRa 904.50 LemTKRP-311)19 APSGFLGTRa 904.50 LemTKRP-4 APSGFMGMRa 952.45 PeaTKRP-4 APGSGFMGMRa 1009.47 LemTKRP-5 APAMGFQGVRa 1032.54 LemTKRP-5 APAMGFQGVRa 1032.54 LemTKRP-6 APAAGFFGMRa 1023.52 PeaTKRP-6 APASGFFGMRa 1039.51 LemTKRP-7 VPASGFFGMRa 1067.55 LemTKRP-7 VPASGFFGMRa 1067.54 LemTKRP-8 GPSMGFHGMRa 1075.49 LemTKRP-8 GPSMGFHGMRa 1075.49 LemTKRP-9 APSMGFQGMRa 1080.51 PeaTKRP-9 APSLGFQGMRa 1062.55 LemTKRP-10 GPNMGFMGMRa 1096.48 PeaTKRP-10 APNMGFMGMRa 1110.50 LemTKRP-11 MGFMGMRa 828.36 LemTKRP-11 MGFMGMRa 828.36 LemTKRP-12 GPSVGFFAMRa 1067.55 LemTKRP-12 GPSVGFFAMRa 1067.55 LemTKRP-13 APSAGFMGMRa 1023.49 LemTKRP-13 APSAGFMGMRa 1023.49 LemTKRP-9 APSMGFQGMRa 1080.51 PeaTKRP-14 APSAGFHGMRa 1029.50
Trang 8N-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
expres-sion 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-transla-tional processing [5]
Previous work has indicated that the different
Lem-TKRPs and the D melanogaster and Locusta
migrato-ria 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
pos-sible that several of the TKRPforms are
physiologi-cally 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
inverte-brates studied so far Most of the 14–15 peptides
pre-dicted 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
Insects
Cockroaches, L maderae and P americana, were raised
under a 12 h light, 12 h dark photoperiod at a constant
temperature of 28C 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
preprotachy-kinin 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 2 Mixture (Clontech, Palo Alto, CA, USA) at low
strin-gency Among the primer combinations tested one pair of
primers amplified approximately 790 bp fragments encoding
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:
lemRACE-B1: 5¢-GTCTACCAAGTCTCGAAGAAAGTCCTGCTG-3¢; peaRACE-F1: 5¢-GATGGAGGGCGCGGAGGAT-5¢-GTCTACCAAGTCTCGAAGAAAGTCCTGCTG-3¢; peaRACE-B1: 5¢-CTTGCCCCTCATGCCATGGAAC-3¢ For both RACE reactions we used Advantage Taq 2 Mixture (Clontech) and a P americana RACE library con-structed previously [39] A L maderae RACE library was prepared from several tissues (brain, ganglia, malpighian tubules, muscles and intestine) using Trizol Reagent (Invi-trogen, Karlsruhe, Germany) for RNA preparation, Oligo-tex (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 prod-ucts 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 par-tially 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 5 lL methanol⁄ water ⁄ trifluoroace-tic acid (90 : 9 : 1, v⁄ v ⁄ v) for electrospray ionization quad-rupole time-of-flight mass spectrometry (ESI-Q-TOF MS)
MALDI-TOF MS
Neurons⁄ glomeruli were dried on the sample plate and sub-sequently rinsed with water to reduce salt contamination Matrix solution (a-cyano-4-hydroxycinnamic acid dissolved
in methanol–water) was pumped onto the dried prepara-tions over a period of approximately 5 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) tri-fluoroacetic acid were added to the 5 lL methanol⁄ water ⁄ trifluoroacetic acid The extract was sonicated, centrifuged
Trang 9and the methanol evaporated from the supernatant The
resulting aqueous supernatant was then loaded onto an
acti-vated and equilibrated home-made micro column
(purifica-tion 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
per-formed using synthetic cockroach peptides
(Pea-pyrokinin-2⁄ 5; SPPFAPRLa ⁄ GGGGSGETSGMWFGPRLa)
ESI-Q-TOF MS
Nanoelectrospray mass spectra were acquired in the
posit-ive-ion mode using the API Qstar Pulsar (Applied
Biosys-tems, 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
col-umn Approximately 1 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
equili-brated 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
pep-tides in MS mode, a collision energy (typically 15–40 V)
was applied The m⁄ z of interest was isolated and
fragmen-ted with the instrument in ‘enhance all’ mode MS⁄ MS data
were acquired over 5 min and manually analyzed
HPLC
Midguts of adult P americana were dissected in insect saline
and shortly rinsed with distilled water before being
trans-ferred to 200 lL methanol–water–trifluoroacetic acid
(90 : 9 : 1, v⁄ v ⁄ v, on ice) Following sonication and
centrifu-gation, 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
SEP-PAK C-18 cartridge (Waters, Milford, MA, USA), pep-tides were eluted with 40% (v⁄ v) acetonitrile containing 0.1% (v⁄ v) trifluoroacetic acid Peptide separation was per-formed on a Shimadzu HPLC system (Shimadzu, Duisburg, Germany) equipped with a diode-array detector and using a Luna RP-C18column (150· 4.6 mm, 100 A˚, 5 lm, Pheno-menex, Torrance, CA, USA) with a linear AB gradient of 10–80% B over 35 min (flow rate: 1 mLÆmin)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 ana-lyzed on a MALDI-TOF mass spectrometer
Immunocytochemistry
Dissected cockroach brains were fixed overnight at 4C 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 5 days at 4C in anti-LomTKRP serum (1 : 1000, diluted with NaCl⁄ Pi1% (v⁄ v) Triton X-100 con-taining 0.25% (w⁄ v) bovine serum albumin and normal goat serum) Following overnight washing in 0.1 molÆL)1 Tris⁄ HCl, 3% (w ⁄ v) NaCl, 1% (v ⁄ v) Triton X-100 (pH 7.6), the fluorochrome-labeled secondary Cy3 antibod-ies were used directly as a mixture in NaCl⁄ Pi–bovine serum albumin (2.5 mgÆmL)1) at a concentration of
1 : 3000 for 4 days Finally, the preparations were washed again overnight in 0.1 molÆL)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 (Euro-mex Microscopes, Arnhem, the Netherlands) Immunostai-nings 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 photoshop7.0 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 abbrevi-ation for the transient receptor potential cabbrevi-ationic channel (TRP) and tryptophan (Trp) Thus the old acronyms such
as LemTRP-1, were changed to LemTKRP-1 The designa-tions of the P americana TKRPs were made such that peptides identical to L maderae peptides are called Lem-TKRPs, 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
Trang 10are given numbers TKRP-10–14 Unique PeaTKRPs are
given numbers that are the same as their LemTKRP
ortho-logs 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
Lem-TKRP-10 identified here is a totally new peptide)
Acknowledgements
We acknowledge the financial assistance of the
Deut-sche Forschungsgemeinschaft (Predel 595⁄ 6–1,2)
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