|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 15, 2007
Journal of Experimental Biology 210, 2278-2289 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004770
Identification and cardiotropic actions of sulfakinin peptides in the American lobster Homarus americanus
1 Department of Biology, Bowdoin College, 6500 College Station, Brunswick,
ME 04011, USA
2 Department of Biology, University of Washington, Box 351800, Seattle, WA
98195-1800, USA
3 Mount Desert Island Biological Laboratory, PO Box 35, Old Bar Harbor Road,
Salisbury Cove, ME 04672, USA
4 School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison,
WI 53705-2222, USA
5 Department of Chemistry, University of Wisconsin, 1101 University Avenue,
Madison, WI 53706-1396, USA
* Author for correspondence (e-mails: pdickins{at}bowdoin.edu)
Accepted 3 April 2007
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: cDNA, neurohormone, pEFDEY(SO3H)GHMRFamide, GGGEY(SO3H)DDY(SO3H)GHLRFamide, heart, sulfakinin, Homarus americanus, expressed sequence tag (EST), neuromodulation, cardiac ganglion
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
Passano, 1951). Likewise, the
neural circuits contained within the crustacean stomatogastric and cardiac
nervous systems have served as two of the premier models for investigating the
generation, maintenance and modulation of rhythmic behavior at the cellular
and systems levels (Selverston and
Moulins, 1987; Harris-Warrick
et al., 1992; Cooke,
2002). Despite their commercial and biological importance,
relatively little is known about these animals at the molecular level. This
lack of information is due, in part, to the scarcity of nucleotide sequence
data for them; e.g. prior to 2006, less than 100 nucleotide sequences were
known from the American lobster Homarus americanus, although it has
been the subject of extensive physiological investigations
(Towle and Smith, 2006).
To help facilitate gene-based studies of crustacean biology, expressed
sequence tags (ESTs) have recently been produced for the normalized cDNA
libraries of several decapod species, including H. americanus
(Towle and Smith, 2006). As
the tissues used to construct some of the libraries include neural and
endocrine tissues, the ESTs generated from them provide useful tools to search
for putative cDNAs encoding peptide precursor proteins. With the
identification of such transcripts, not only can the amino acid sequences of
the encoded peptide hormones be deduced, but their distributions and
regulation within neural and endocrine systems can also be investigated. To
this end, we have recently begun database mining of several H.
americanus EST collections for prepro-hormone transcripts, particularly
those that encode neuropeptides that are likely present in the nervous system
in low abundance and/or ones with modifications that make them difficult to
identify using other methods.
One family of peptides that has long proved challenging to identify in
decapod crustaceans is the sulfakinins, a group of peptides containing the
carboxy (C)-terminal motif –Y(SO3H)GHM/LRFamide. First using
biochemical methods, and more recently via molecular techniques, a
number of sulfakinin isoforms have been identified from insects
(Nachman et al., 1986a;
Nachman et al., 1986b;
Nichols et al., 1988;
Veenstra, 1989;
Schoofs et al., 1990;
Fonagy et al., 1992;
Nichols, 1992;
Duve et al., 1995;
Maestro et al., 2001) (NCBI
accession number AY341429; NCBI accession number AY758365)
(Table 1). In contrast,
sulfakinin isoforms have thus far been biochemically isolated and
characterized from just two crustaceans, the penaeid shrimp Penaeus
monodon and Litopenaeus vannamei
(Johnsen et al., 2000;
Torfs et al., 2002)
(Table 1). For each shrimp
species, over a thousand central nervous systems (CNSs) [1030 for P.
monodon (Johnsen et al.,
2000) and 3500 for L. vannamei
(Torfs et al., 2002)] were
needed to isolate and characterize the native sulfakinins,
pQFDEY(SO3H)GHMRFamide and
AGGSGGVGGEY(SO3H)DDY(SO3H)GHLRFamide.
|
Undoubtedly, large tissue pools were necessary for these identifications
due, in part, to the fact that the shrimp CNS possesses a very small number of
sulfakinin-containing neurons [10 or fewer in P. monodon
(Johnsen et al., 2000), a
distribution shared with insects (Duve et
al., 1994; Davis et al.,
1996; Nichols and Lim,
1996; East et al.,
1997)]. As such, large tissue samples are impractical to collect
from H. americanus, we have taken advantage of H. americanus
ESTs to identify and sequence a cDNA encoding prepro-sulfakinin from this
species. Based on modifications suggested by peptide modeling programs and on
homology to the known sulfakinins, particularly those from shrimp
(Johnsen et al., 2000;
Torfs et al., 2002), the
putative mature forms of the H. americanus sulfakinins were predicted
and the peptides synthesized. Exogenous application of each of the predicted
peptides to the lobster heart produced dramatic increases in both contraction
amplitude and frequency, suggesting that the hypothesized post-translational
modifications were correct and demonstrating that each isoform is bioactive on
the heart. Taken collectively, our data provide the first molecular
characterization of a sulfakinin-encoding cDNA from a crustacean and
demonstrate, for the first time, that native sulfakinins are biologically
active in this group of arthropods. Some of these data have appeared
previously in abstract form (Brennan et
al., 2006).
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
cDNA library construction, normalization, sequencing and EST submission
The construction and normalization of the H. americanus cDNA
library used in this study were described in detail in a previous report
(Towle and Smith, 2006). In
brief, multiple tissues (including the supraoesophageal ganglion, commonly
referred to as the brain) from four individuals were collected, total RNA
samples were prepared individually from each tissue, checked for quality, then
pooled for construction and normalization of a cDNA library by Invitrogen
Corporation (Carlsbad, CA, USA). Plasmids were isolated and inserts
single-pass sequenced from their 5' end using SP6 primer
(5'-ATTTAGGTGACACTATAG-3') at the Marine DNA Sequencing and
Analysis Facility at Mount Desert Island Biological Laboratory (Salisbury
Cove, ME, USA). Sequence traces were processed for submission to dbEST
(National Center for Biotechnology Information; Bethesda, MD, USA) using the
trace2dbest component of PartiGene software (University of Edinburgh,
Edinburgh, Scotland, UK). Before submission, all ESTs were subjected to blastx
analysis [i.e. translated nucleotide sequence versus protein sequence
(Altschul et al., 1997)] and
annotated accordingly. A single EST (NCBI accession number CN952349) with
significant homology to a sulfakinin precursor from the blowfly Calliphora
vomitoria (NCBI accession number Q7M3V5)
(Duve et al., 1995) was
identified during this analysis.
cDNA sequence analysis
To characterize the H. americanus cDNA clone (Ha_mx0_58h07)
identified by blastx analysis, a sample of the bacteria (Escherichia
coli) possessing the insert-containing vector was cultured overnight in
LB-medium at 37°C. Plasmid containing the cDNA was subsequently isolated
using a PurelinkTM Quick Plasmid Miniprep kit (Invitrogen). The vector
insert was then sequenced on an ABI 3100 16-capillary sequencer (Applied
Biosystems Incorporated, Foster City, California, USA) using both vector- and
insert-specific forward and reverse sequencing primers (Integrated DNA
Technologies, Inc., Coralville, IA, USA;
Table 2). The sequence trace
files resulting from each round of sequencing were analyzed using Chromas 2.31
software (Technelysium Pty Ltd, Tewantin, Queensland, Australia), and the high
quality nucleotide sequences were aligned using SeqMan 2.6 software (DNASTAR
Inc., Madison, WI, USA).
|
Nucleotide translation and structural analysis of the deduced amino acid sequence
Translation of the full-length nucleotide sequence of cDNA clone
Ha_mx0_58h07 was accomplished using the online program WWW Nucleotide
Translation (BioInformatics & Molecular Analysis Section [BIMAS], National
Institutes of Health, Bethesda, MD, USA;
http://bimas.dcrt.nih.gov/molbio/translate/).
Signal peptide and signal peptide cleavage prediction was done via
the online program SignalP 3.0 using both Neural Networks and Hidden Markov
Models algorithms (Center for Biological Sequence Analysis, Technical
University of Denmark, Lyngby, Denmark;
http://www.cbs.dtu.dk/services/SignalP/)
(Bendtsen et al., 2004).
Prohormone cleavage sites were predicted based on the information presented in
several recent reviews (Veenstra,
2000; Fricker,
2005). Prediction of the sulfation state of tyrosine residues was
done using the online program Sulfinator (Swiss Institute of Bioinformatics,
Geneva, Switzerland;
http://www.expasy.org/tools/sulfinator/)
(Monigatti et al., 2002), as
well as through homology to known sulfakinin sequences from shrimp
(Johnsen et al., 2000;
Torfs et al., 2002). Likewise,
other post-translational modifications (i.e. cyclization of N-terminal
glutamic acid residues and C-terminal amidations) were predicted by homology
to known sulfakinin sequences, particularly those isolated from shrimp
(Johnsen et al., 2000;
Torfs et al., 2002).
Production of synthetic peptides
pEFDEY(SO3H)GHMRFamide and
GGGEY(SO3H)DDY(SO3H) GHLRFamide were synthesized on an
ABI Pioneer peptide synthesizer (Applied Biosystems Inc.) using standard Fmoc
chemistry at the Biotechnology Center of the University of Wisconsin-Madison
(Madison, WI, USA). Fmoc-Tyr(SO3-OH) sodium salt was purchased from
Chem-Impex (Wood Dale, IL, USA). The remaining Fmoc-amino acids, including
pyroglutamine, were purchased from Novabiochem (San Diego, CA, USA). For the
synthesis of pEFDEY(SO3H) GHMRFamide, a 60 min extended coupling
time was used to couple the sulfotyrosine residue and double 30 min couplings
were used for the three N-terminal amino acids following the sulfotyrosine.
Coupling times were 30 min for the first five C-terminal amino acids. For the
synthesis of GGGEY(SO3H) DDY(SO3H)GHLRFamide, a 60 min
extended coupling time was used for both sulfotyrosine couplings and double 30
min couplings were used for the two aspartic acid couplings following the
first sulfotyrosine and for all four N-terminal residues following the second
sulfotyrosine. Coupling times were 30 min for the first five C-terminal amino
acids.
Following the completion of all coupling reactions, pEFDEY(SO3H)GHMRFamide and GGGEY(SO3H)DDY(SO3H) GHLRFamide were cleaved and deprotected for 90 min in thioanisole: ethanedithiol: trifluoroacetic acid (TFA) (5: 2.5: 92.5). The resin was then filtered off and the cleavage solution for each synthesis was dripped into 10 ml of cold t-butylmethyl ether to precipitate the peptide. The resulting precipitates were washed and centrifuged three times with additional volumes of ether. The ether precipitates were then dried by vacuum. To avoid acid hydrolysis of the sulfate groups, the crude peptides were dissolved in water:ammonium hydroxide (100:1) to neutralize the residual TFA.
Crude pEFDEY(SO3H)GHMRFamide and GGGEY(SO3H) DDY(SO3H)GHLRFamide were purified on a Beckman System Gold HPLC system (Fullerton, CA, USA) using a preparative C-18 reverse phase column (Dynamax 250x21.4 mm; Varian, Palo Alto, CA, USA). Solvent A was 0.1% TFA/water and Solvent B was 0.08% TFA/90% acetonitrile. The gradient program was 0% B (0–8 min), 0–7% B (8–11 min), 11–26% B (11–77 min), with a flow rate was 16 ml min-1. Each collected fraction (9.6 ml) was neutralized immediately by addition of 20 µl of concentrated ammonium hydroxide to prevent the hydrolytic loss of sulfate groups. The final purities of pEFDEY(SO3H)GHMRFamide and GGGEY(SO3H)DDY(SO3H) GHLRFamide were 94% and 90%, respectively.
Cardiac physiology
To determine the effects of the sulfakinins on the neurogenic heart of
H. americanus, lobsters were cold-anaesthetized by packing them in
ice for 30–60 min, after which the posterior dorsal region of the
thoracic carapace that lies directly over the heart, as well as the underlying
cardiac tissue, was removed. This dissected region was pinned through the
carapace to the bottom of a small Sylgard 184 (KR Anderson, Santa Clara, CA,
USA)-lined dish. The dorsal part of the heart remained attached to the
carapace, so that the extent to which it was stretched was identical to that
in the intact animal. The posterior artery was cannulated with a short piece
of polyethylene tubing drawn out to fit the artery, and was continuously
perfused with physiological saline [composition in mmol l-1: 479.12
NaCl, 12.74 KCl, 13.67 CaCl2, 20.00 MgSO4, 3.91
Na2SO4, 5.00 Hepes, pH 7.4
(Bucher et al., 2003)] cooled
with a Warner Instruments CL-100 bipolar temperature control system (Hamden,
CT, USA). Because isolated hearts continue to contract only when adequately
stretched (Cooke, 2002), flow
rate through the heart was kept at approximately 2 ml min-1. Under
our recording conditions, stable heart activity could be recorded for at least
8 h. A second perfusion line was directed across the top of the heart to help
maintain temperature, which was monitored continuously and kept between
10–12°C.
To record heart contractions, the anterior arteries were tied off with a human hair and attached to a Grass FT03 force-displacement transducer (Astro-Med, Inc., West Warwick, RI, USA) at an angle of approximately 30°. The output of the transducer was amplified via a Brownlee 410 instrumentation amplifier (San Jose, CA, USA), and recorded onto a PC computer using a Micro 1401 data acquisition board and Spike2 version 5 software (Cambridge Electronic Design Limited, Cambridge, UK). Both heart rate and contraction amplitude were measured using the built-in functions of Spike2. Data were further analyzed and graphed using Prism4 software (GraphPad Software, Inc., San Diego, CA, USA).
Preparations were allowed to stabilize for 1–2 h before the first application of sulfakinin. Both Hoa-SK I and Hoa-SK II were dissolved in deionized water at a concentration of 10–3 mol l-1, and kept as a frozen stock solution at –20°C for use in physiological experiments. Control experiments to compare the effects of frozen stock peptide and freshly dissolved peptide showed that both sulfakinin isoforms were stable when frozen in aqueous solution (data not shown). Peptides were diluted in chilled physiological saline to a final concentration of 10–6 mol l-1 just before use. Both Hoa-SK I and II were applied to most preparations, in random order, with at least 1 h of wash in control saline between peptides, to allow for comparisons within the same preparation. No order effect was seen (data not shown).
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
) was
identified. Using a combination of vector- and insert-specific forward- and
reverse-sequencing primers (Table
2), a 1523 base pair (bp), putative full-length cDNA was sequenced
(NCBI accession number EF418605). As is shown in
Fig. 1, this full-length clone
consisted of a 42 bp 5'-untranslated region (UTR), a 363 bp open-reading
frame (ORF), as well as a 1118 bp 3'-UTR containing two AATAAA
polyadenylation signal sequences located 12 and 658 bps upstream of a 79 bp
poly-A tail.
|
) of this
amino acid sequence using both Neural Networks and Hidden Markov Models
algorithms identified the first 24 amino acids of the prepro-hormone as a
signal peptide, with a cleavage site between Ser24 and
Ala25 (Figs 1 and
2). Processing between these
residues would produce a 96 aa pro-sulfakinin that contains two Lys-Arg and
two Arg-Xn-Arg (where X is a variable amino acid and n is either 2
or 6) processing sites (Veenstra,
2000; Fricker,
2005) (Figs 1 and
2). Among the peptides
predicted to be cleaved from the pro-peptide via proteolytic
processing are EFDEYGHMRFG (Figs
1 and
2) and GGGEYDDYGHLRFG (Figs
1 and
2), both of which contain the
core sequence –YGHM/LRF that is the hallmark of the sulfakinin family
(Table 1). In addition to the
sulfakinins, three other peptides, collectively termed sulfakinin
precursor-related peptides (SPRPs), are also predicted to be liberated from
the pro-hormone: APARPSSLARVLAPVV (SPRP I; Figs
1 and
2,
Table 1),
QRLEESHLPPALVEELVQDFEDPELLDFHDAAG (SPRP II; Figs
1 and
2,
Table 1) and SLTHSDQHHHHDTTVN
(SPRP III; Figs 1 and
2,
Table 1).
|
;
Torfs et al., 2002), it is
likely that the immature H. americanus sulfakinins EFDEYGHMRFG and
GGGEYDDYGHLRFG undergo significant post-translational modification prior to
assuming their mature, bioactive isoforms
(Fig. 2,
Table 1). Specifically, the
C-terminal glycine residue of each peptide is predicted to act as a donor for
amidation in the mature peptides, and the N-terminal glutamic acid of
EFDEYGHMRFG is predicted to become cyclized to pyroglutamic acid
(Fig. 2). The tyrosine residue
or residues contained within each of the predicted immature sulfakinins are
also likely modified by the addition of sulfate groups; however, the two
previous studies in shrimp (Johnsen et
al., 2000; Torfs et al.,
2002) provided conflicting pictures as to whether one or both of
the tyrosines in GGGEYDDYGHLRFG are sulfated. In P. monodon, only
Tyr14 was identified as sulfated in AGGSGGVGGEYDDYGHLRFamide
(Johnsen et al., 2000), while
in L. vannamei both Tyr11 and Tyr14 were
identified as sulfated in this peptide
(Torfs et al., 2002). Torfs et
al. (Torfs et al., 2002)
suggested that the discrepancy in the sulfation states of the peptides was due
to the different methods used to assess the presence of this modification,
i.e. MALDI-TOF MS and immunohistochemistry with sulfate-specific antisera [for
P. monodon (Johnsen et al.,
2000)] versus electrospray ionization MS and bioassay
[for L. vannamei (Torfs et al.,
2002)], with the latter combination of techniques providing a more
accurate picture of the extent of tyrosine sulfation. Analysis of the H.
americanus sulfakinin sequences using the Sulfinator software program
(Monigatti et al., 2002)
predicted all tyrosine residues in both isoforms are likely to be sulfated (E
values <55 for each residue). Thus, we agree with the hypothesis put forth
by Torfs et al. (Torfs et al.,
2002) and predict the mature H. americanus sulfakinin
isoforms to have the following structures: pEFDEY(SO3H) GHMRFamide
and GGGEY(SO3H)DDY(SO3H)GHLRFamide
(Fig. 2,
Table 1). Based on standard
convention, we named these peptides Homarus americanus sulfakinin I
(Hoa-SK I) and Homarus americanus sulfakinin II (Hoa-SK II),
respectively.
In addition to the sulfakinin isoforms, post-translational processing is also predicted for at least one of the encoded SPRPs, specifically Hoa-SPRP II. As with the sulfakinin isoforms, the C-terminal glycine residue is likely converted to an amide group, producing the peptide QRLEESHLPPALVEELVQDFEDPELLDFHDAAamide(Fig. 2, Table 1). It is also possible that the N-terminal glutamine in Hoa-SPRP II is enzymatically cyclized to pyroglutamic acid. Thus, the mature form of Hoa-SPRP may be either QRLEESHLPPALVEELVQDFEDPELLDFHDAAamide or pQRLEESHLPPALVEELVQDFEDPELLDFHDAAamide (Fig. 2, Table 1). No obvious motifs for post-translational processing are present in either Hoa-SPRP I or III and thus we predict their mature isoforms to be APARPSSLARVLAPVV and SLTHSDQHHHHDTTVN, respectively (Fig. 2, Table 1).
Physiological effects of Hoa-SK I and II on the heart
Based on our collective molecular and bioinformatic data, the mature forms
of Hoa-SK I and Hoa-SK II were predicted and synthesized. To assess their
potential bioactivity in H. americanus, we applied them to the
isolated neurogenic heart, a known target of many circulating peptide hormones
(Cooke, 2002). Under control
conditions, both the heart rate and the specific pattern of cardiac
contractions recorded in isolated Homarus hearts varied somewhat,
with frequencies ranging from an average of approximately 0.2 Hz to 1.0 Hz. In
approximately 80% of preparations, the heartbeat was extremely regular
(Fig. 3A,B), while in the
remaining 20%, the heartbeat was regularly interrupted by a sequence (usually
4–6) of rapid and small amplitude beats followed by a long interbeat
interval (Fig. 3C,D). In the
examples shown in Fig. 3, it
can be seen that upon application of either Hoa-SK I or Hoa-SK II, the
heartbeat frequency increased in both types of preparations, as is
particularly clear in the response to Hoa-SK II. When data from a number of
preparations were pooled (Fig.
4), it was seen that exogenous application of both Hoa-SK I and
Hoa-SK II (10–6 mol l-1) was strongly
cardioexcitatory, increasing the frequency of spontaneous contractions by
14.9±5.5% (± s.e.m.) and 19.4±5.7%, respectively.
Calculated percent changes were significantly different from zero in both
peptides (two-tailed single sample t-test: Hoa-SK I frequency,
P<0.05; Hoa-SK II frequency, P<0.01; N=11
preparations for Hoa-SK I, N=14 preparations for Hoa-SK II). In
addition, both Hoa-SK I and Hoa-SK II induced a regularization of the
heartbeat in all preparations in which an irregular beat was present in
control saline, as seen in Fig.
3C,D. In both peptides, regardless of the extent to which the
heartbeat increased in frequency, the rapid beats and long interbeat intervals
were eliminated in the presence of the peptide. The heartbeat again became
irregular in the wash with physiological saline.
|
|
In addition to changes in the heartbeat frequency, both Hoa-SK I and Hoa-SK II caused significant increases in contraction amplitude of the heart, as can be seen in both the individual traces in Fig. 3B,D and in the pooled data in Fig. 4. Hoa-SK I increased amplitude by 15±4.4% (± s.e.m.) over control values, while Hoa-SK II caused an increase of 25.3±4.5% over control (single sample t-test; values significantly different from 0; P<0.01, N=11 preparations in Hoa-SK I and N=14 preparations in Hoa-SK II).
The effects of both Hoa-SK I and Hoa-SK II on heart contraction frequency were rapidly reversible, returning to control levels after approximately 15–20 min of superfusion with physiological saline (Fig. 5). The effects of the two peptides on contraction amplitude followed a very similar time course (compare Fig. 5A with 5C and 5B with 5D).
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
; Nachman et al.,
1986b): EQFDEDY(SO3H)GHMRFamide and
pQSDDY(SO3H)GHMRFamide from the cockroach Leucophaea
maderae. This sulfation, in combination with their strong myotropic
activity on the hindgut, resulted in these peptides being named sulfakinins
(Nachman et al., 1986a;
Nachman et al., 1986b). In the
years that have followed, additional sulfakinins have been isolated
biochemically and the prepro-hormones encoding members of this peptide family
have been isolated. By far the majority of these peptides have been identified
in insects (Nachman et al.,
1986a; Nachman et al.,
1986b; Nichols et al.,
1988; Veenstra,
1989; Schoofs et al.,
1990; Fonagy et al.,
1992; Nichols,
1992; Duve et al.,
1995; Maestro et al.,
2001) (NCBI accession number AY341429; NCBI accession number
AY758365) (Table 1). In
addition, however, sulfakinins have been isolated from two species of penaeid
shrimp, Penaeus monodon and Litopenaeus vannamei
(Johnsen et al., 2000;
Torfs et al., 2002)
(Table 1), demonstrating the
conservation of this peptide group across the arthropods. In the present study
we report the identification of the first cDNA encoding sulfakinins from a
crustacean, namely one from the American lobster H. americanus.
Comparisons of Hoa-prepro-sulfakinin to its insect homologs
(Nichols et al., 1988;
Duve et al., 1995) (NCBI
accession number AY341429; NCBI accession number AY758365) show a number of
conserved features. Specifically, all known prepro-hormones encode two
distinct sulfakinin sequences, one short and one long, e.g. EFDEYGHMRF and
GGGEYDDYGHLRF in H. americanus (this study) and FDDYGHMRF and
GGEEQFDDYGHMRF in the blowflies C. vomitoria and Lucilia
cuprina (Duve et al.,
1995). In all species, the short form is preceded by the dibasic
cleavage sequence KR and precedes the long form within the prepro-hormone.
Likewise, in all known prepro-sulfakinins, the short form is separated from
the long form by the amidating cleavage sequence GKR, with the long form being
followed by the amidating cleavage sequence GR. Interestingly, while both the
short and long isoforms are present in the same copy number within the
prepro-hormones, in species in which the peptides have been biochemically
isolated, there appears to be a much higher abundance of the shorter peptide
(e.g. Nichols et al., 1988;
Nichols, 1992;
Duve et al., 1995;
Johnsen et al., 2000;
Torfs et al., 2002). Thus, it
may be that prohormone convertase preferentially targets the dibasic cleavage
sequence over the monobasic site, though differential degradation rates by
peptidases may also play a role in controlling the relative abundance of the
isoforms.
In addition to their encoded sulfakinin isoforms, all prepro-sulfakinins
also possess other amino acid sequences that are predicted to be cleaved from
them, producing a number of other peptides, termed here sulfakinin
precursor-related peptides or SPRPs
(Nichols et al., 1988;
Duve et al., 1995) (NCBI
accession number AY341429; NCBI accession number AY758365; this study). In
H. americanus, these sequences are APARPSSLARVLAPVV,
QRLEESHLPPALVEELVQDFEDPELLDFHDAAG and SLTHSDQHHHHDTTVN. Unlike the sulfakinin
sequences, limited conservation is present in the SPRP sequences across
species and no functional roles have been ascribed for these peptides in any
species, if they are indeed bioactive.
Sulfakinin isoforms appear highly conserved both within and between taxa
As is the case in other species, two sulfakinins, one short and one long
isoform, are encoded in the predicted H. americanus prepro-hormone.
Based on bioinformatics and homology to known sulfakinin sequences
(Table 1), we predicted the
mature H. americanus structures to be
pEFDEY(SO3H)GHMRFamide (Hoa-SK I) and
GGGEY(SO3H)DDY(SO3H)GHLRFamide (Hoa-SK II). If our
predictions are correct, Hoa-SK I would be identical to a previously described
shrimp isoform, i.e. Pem-SK I/Pev-SK II
(Johnsen et al., 2000;
Torfs et al., 2002)
(Table 1), while Hoa-SK II
would be a novel sulfakinin family member
(Table 1). It should be noted
that the N-terminal pyro-residue of Pem/Pev-SK I is given as being derived
from glutamine rather than glutamic acid (as we have shown here is the case
for Hoa-SK I), though there are no data to prove unambiguously whether the
cyclization is truly derived from this amino acid.
It is interesting to note that the short, but not the long, sulfakinin
isoform is identically conserved between lobster and shrimp, which represent
two distinct, and rather distantly related infraorders of the decapods
(Johnsen et al., 2000;
Torfs et al., 2002) (this
study, Table 1). While the
functional significance of a conserved short isoform and a variable long
isoform is presently unknown, similar patterns of conservation have been noted
in related insect taxa (Table
1). For example, FDDY(SO3H)GHMRFamide has been
identified or is predicted to be present in each of the dipteran (true fly)
species thus far studied, whereas the longer sulfakinin isoform varies
considerably between species (Nichols et
al., 1988; Fonagy et al.,
1992; Duve et al.,
1995) (NCBI accession number AY341429; NCBI accession number
AY758365) (Table 1). Similarly,
pQSDDYGHMRFamide is shared by the two blattodean (cockroach) species thus far
examined, while their extended isoforms differ
(Nachman et al., 1986a;
Nachman et al., 1986b;
Veenstra, 1989)
(Table 1). As additional
studies are conducted on other crustacean and hexapod species, it will be
interesting to see if this pattern of conservation holds as a rule or whether
it is simply a function of not yet having a broad enough picture of the extant
sulfakinin family members. If the former, extensive physiological
investigations of sulfakinin actions on multiple tissues, as well as the
identification and functional studies on the sulfakinin receptor(s) will be
needed to elucidate the selective pressures resulting in the differential
conservation of the short versus long sulfakinin isoforms.
Hoa-SK II, along with the long sulfakinin isoforms isolated previously from
penaeid species (Johnsen et al.,
2000; Torfs et al.,
2002), possess several structural features that distinguish them
from all other members of the sulfakinin family
(Table 1). First, Hoa-SK II and
Pem-SK II/Pev-SK I each end in –LRFamide rather than the usual
–MRFamide C terminus. Moreover, these peptides contain two, rather than
one, tyrosine residue, both of which are likely to be sulfated. Finally, the
peptides contain glycine-rich N-termini, with the shrimp isoform exhibiting
this feature to a greater extent than Hoa-SK II. The functional significance
of these features is presently unknown, though as discussed below, they may
well manifest themselves functionally in at least H. americanus, as
Hoa-SK II appears to be a stronger modulator of the cardiac system than is
Hoa-SK I.
The sulfakinins appear to be multifunctional peptides
Physiological investigations in insects have shown the sulfakinins to be
multifunctional. Their discovery in the cockroach L. maderae was
based on the hindgut myotropic assay
(Nachman et al., 1986a;
Nachman et al., 1986b), and
they appear to possess similar function in most
(Schoofs et al., 1990;
Predel et al., 1999) but not
all species, i.e. the blowfly C. vomitoria
(Duve et al., 1994;
Duve et al., 1995). In the
cockroach P. americana, the native sulfakinins have been shown to be
cardioactive (Predel et al.,
1999). While not demonstrated directly, the distributions of
sulfakinin-like immunoreactivity in many insects, particularly the dipterans,
suggests a neuromodulatory role for this peptide family in the CNS
(Duve et al., 1994;
Davis et al., 1996;
Nichols and Lim, 1996;
East et al., 1997).
Additionally, sulfakinins have been shown to stimulate the release of
-amylase from the midgut of both the weevil Rhynchophorus
ferrugineus and the moth Opisina arenosella
(Nachman et al., 1997;
Harshini et al., 2002), and
have been shown to be potent inhibitors of food intake in the locust
Schistocerca gregaria, the cockroach Blatella germanica and
the blowfly Phormia regina (Wei
et al., 2000; Maestro et al.,
2001; Downer et al.,
2007).
Prior to our study, the functional roles played by sulfakinins in
crustaceans were unknown. As in the dipterans, mapping of the distribution of
sulfakinin-like labeling in P. monodon suggested a neuromodulatory
role for family members in the shrimp CNS
(Johnsen et al., 2000). Both
the stomatogastric nervous system and the cardiac ganglion system have served
as excellent models for the study of neuromodulation in decapods. Although we
are currently investigating the effects of sulfakinins on the pattern
generators of the stomatogastric system, the effects on these pattern
generators in preliminary experiments appear to be relatively weak, so we have
chosen to focus in this study on the effects of sulfakinins on the heartbeat
of the lobster. Thus, in this study, we have shown that both of the native
isoforms of sulfakinin are potent modulators of heartbeat in vitro.
Specifically, exogenous application of either peptide to the isolated heart
increased both the frequency and amplitude of spontaneous heart contractions.
Moreover, both peptides regularized the pattern of spontaneous contractions in
preparations where the heartbeat was initially irregular. Irregularities in
crustacean heartbeat frequency and amplitude have been noted in a number of
previous studies, or are seen in the recordings shown in these studies
(Kuramoto and Ebara, 1984;
Kuramoto and Ebara, 1985;
Mercier and Russenes, 1992;
Hokkanen, 2000). Kuramoto and
Ebara (Kuramoto and Ebara,
1985) noted that both heartbeat frequency and the variability in
heartbeat parameters are influenced by the perfusion pressure. Worden et al.
(Worden et al., 2006) also
noted that heartbeat frequency varies significantly as a function of
temperature. In the present study, both perfusion pressure and temperature
were held constant during each experiment, and the peptide alone was
responsible for changing the irregular heartbeat to a regular one. We do not
yet know what mechanisms may underlie either the initial irregularities or the
regularization that occurred in response to sulfakinin application, but future
examinations of the effects of the peptides on the output of the cardiac
ganglion itself may help to elucidate these issues.
While both Hoa-SK I and Hoa-SK II exerted similar actions on the heart qualitatively, Hoa-SK II was more potent than Hoa-SK I on all examined effects. In terms of their ability to modulate heartbeat frequency, Hoa-SK II was approximately 5% more potent then Hoa-SK I in increasing the frequency of spontaneous contractions over controls. Likewise, it was approximately 10% more potent than Hoa-SK I in effecting an increase in contraction amplitude. At present, the mechanism(s) by which these quantitative differences are achieved is unknown. Moreover, we have not yet examined the thresholds for the effects of these peptides, which might also differ. These experiments, and experiments designed to determine whether the specific effects exerted by the sulfakinins are dose-dependent, are ongoing. Clearly the structures of the two peptides are distinct. Most notably, Hoa-SK II is a disulfated peptide whereas Hoa-SK I contains only a single sulfated tyrosine residue. Hoa-SK II also possesses a leucine for methionine substitution in its C terminus that is not present in Hoa-SK I, and Hoa-SK II is longer than Hoa-SK I: 13 versus 10 amino acids in total length, respectively. Any or all of these structural differences might result in differential affinities to bind to a common receptor. Alternatively, the two peptides may target distinct receptors, which results in the quantitatively distinct effects on the heart. It is also possible that the two peptides are differentially sensitive to peptidase actions within the heart tissue and that this results in Hoa-SK II being present at a higher absolute concentration at the receptor(s) than is Hoa-SK I.
Do the invertebrate sulfakinins and the vertebrate CCK/gastrins share a common ancestry?
As has been noted by many authors, the sulfakinins share structural
similarities to members of the vertebrate cholecystokinin (CCK)/gastrin family
of peptides (Table 1), as well
as to cionin (Table 1), a
disulfated peptide isolated from the protochordate tunicate Ciona
intestinalis (Johnsen and Rehfeld,
1990). It is the belief of many that these structural similarities
are the result of a common ancestry for the two groups of peptides
(Nachman et al., 1986a;
Nachman et al., 1986b;
Nichols et al., 1988;
Veenstra, 1989;
Schoofs et al., 1990;
Maestro et al., 2001; Torfs et
al., 2001; Nachman et al.,
2005). In addition to the observed sequence homologies, the
hypothesis of a common ancestor for the CCK/gastrins and the sulfakinins is
supported by the findings that both share a number of conserved functions:
both groups are myoactive on the gut, induce the release of the enzyme amylase
from the digestive system, and serve as satiety factors
(Nachman et al., 1997;
Wei et al., 2000;
Maestro et al., 2001;
Harashini et al., 2002; Downer et al.,
2007). Moreover, studies of the effects of vertebrate CCK and of a
partially purified CCK-like peptide in the spiny lobster showed that CCK
itself can activate the neuronal pattern generators that control movements of
the foregut in this species (Turrigiano
and Selverston, 1989;
Turrigiano and Selverston,
1990; Turrigiano et al.,
1994). The threshold for CCK effects was quite high in these
studies, suggesting that the native peptide is not authentic CCK, but a
related peptide, such as a sulfakinin. One possibility is that the observed
effects of CCK were due to the activation of sulfakinin receptors by CCK;
however, this remains to be tested.
Analyses of the D. melanogaster genome also suggested that the
Drosophila G-protein coupled receptors CG6857 and CG6881 and the
vertebrate CCK/gastrin receptors CCKR and GASR diverged from a common ancestor
(Hewes and Taghert, 2001).
Moreover, the cloning and expression of CG6881 (also known as DSK-R1) in a
mammalian cell line showed it to be the target of a variety of sulfakinin
isoforms (Kubiak et al.,
2002). It should be noted, however, that other authors have
suggested that the convergent evolution of separate lineages, rather than a
shared ancestry, is the source of the structural and functional homologies
seen between the CCK/gastrins and the sulfakinins
(Duve et al., 1994;
Duve et al., 1995;
Johnsen, 1998;
Johnsen et al., 2000). It is
clear that more studies, encompassing a broader sampling of species, like ours
here, will be needed to address this issue fully.
Conclusion
In summary, we have identified and characterized a cDNA from the American
lobster H. americanus encoding a prepro-sulfakinin peptide, which is
the first description of a sulfakinin-encoding cDNA from any crustacean
species. Using bioinformatics and homology to known sulfakinin isoforms, we
predicted the structures of the mature sulfakinins encoded in the
prepro-hormone and synthesized them. Exogenous application of either of the
two native peptides to the isolated H. americanus heart induced both
increased frequency and amplitude of the heartbeat and regularized the rhythm
of contractions in preparations where periodic interruptions were initially
present. Our identification of the native sulfakinins from the lobster opens
the door for future functional studies of these peptides in H.
americanus, and lays a foundation for future comparative studies of the
sulfakinins and their actions in other crustacean species.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Bendtsen, J. D., Nielsen, H., von Heijne, G. and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340,783 -795.[CrossRef][Medline]
Bliss, D. E. (1951). Metabolic effects of sinus gland or eyestalk removal in the land crab, Gecarcinus lateralis.Anat. Rec. 111,502 -503.
Brennan, H. R., Goiney, C. C., Hsu, Y. A., Smith, C. M., Towle, D. W., Dickinson, P. S. and Christie, A. E. (2006).Identification of prepro-sulfakinin in the American lobster Homarus americanus . Program No. 129.5. 2006 Neuroscience Meeting Planner. Atlanta, GA: Society for Neuroscience. Online.
Bucher, D., Thirumalai, V. and Marder, E. (2003). Axonal dopamine receptors activate peripheral spike initiation in a stomatogastric motor neuron. J. Neurophysiol. 23,6866 -6875.
Cooke, I. M. (2002). Reliable, responsive
pacemaking and pattern generation with minimal cell numbers: the crustacean
cardiac ganglion. Biol. Bull.
202,108
-136.
Davis, N. T., Homberg, U., Teal, P. E., Alstein, M., Agricola, H. J. and Hildebrand, J. G. (1996). Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: immunoreactivities to PBAN and other neuropeptides. Microsc. Res. Tech. 35,201 -229.[CrossRef][Medline]
Downer, K. E., Haselton, A. T., Nachman, R. J. and Stoffolano, J. G., Jr (2007). Insect satiety: sulfakinin localization and the effects of drosulfakinin on protein and carbohydrate ingestion in the blow fly Phormia regina (Diptera: Calliphoridae). J. Insect Physiol. 53,106 -112.[CrossRef][Medline]
Duve, H., Rehfeld, J. F., East, P. and Thorpe, A. (1994). Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria. Cell Tissue Res. 275,177 -186.[CrossRef][Medline]
Duve, H., Thorpe, A., Scott, A. G., Johnsen, A. H., Rehfeld, J. F., Hines, E. and East, P. D. (1995). The sulfakinins of the blowfly Calliphora vomitoria. Peptide isolataion, gene cloning and expression studies. Eur. J. Biochem. 232,633 -640.[Medline]
East, P. D., Hales, D. F. and Cooper, P. D. (1997). Distribution of sulfakinin-like peptides in the central and sympathetic nervous system of the American cockroach, Periplaneta americana (L.) and the field cricket, Teleogryllus commodus (Walker). Tissue Cell 29,347 -354.[CrossRef][Medline]
Fonagy, A., Schoofs, L., Proost, P., Van Damme, J. and De Loof, A. (1992). Isolation and primary stucture of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata.Comp. Biochem. Physiol. 103C,135 -142.
Fricker, L. D. (2005). Neuropeptide processing enzymes: applications for drug discovery. AAPS J. 7,E449 -E455.[CrossRef][Medline]
Harshini, S., Nachman, R. J. and Sreekumar, S. (2002). In vitro release of digestive enzymes by FMRFamide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.). Peptides 23,1759 -1763.[CrossRef][Medline]
Harris-Warrick, R. M., Marder, E., Selverston, A. I. and Moulins, M. (ed.) (1992). Dynamic Biological Networks: The Stomatogastric Nervous System. Cambridge: MIT Press.
Hewes, R. S. and Taghert, P. H. (2001).
Neuropeptides and neuropeptide receptors in the Drosophila
melanogaster genome. Genome Res.
11,1126
-1142.
Hokkanen, J. E. (2000). Chaotic or periodic variation? Looking at crustacea hearts. J. Theor. Biol. 203,451 -454.[CrossRef][Medline]
Johnsen, A. H. (1998). Phylogeny of the cholecystokinin/gastrin family. Front. Neuroendocrinol. 19,73 -99.[CrossRef][Medline]
Johnsen, A. H. and Rehfeld, J. F. (1990).
Cionin: a disulfotyrosyl hybrid of cholecystokinin and gastrin from the neural
ganglion of the protochordate Ciona intestinalis. J. Biol.
Chem. 265,3054
-3058.
Johnsen, A. H., Duve, H., Davey, M., Hall, M. and Thorpe, A. (2000). Sulfakinin neuropeptides in a crustacean. Isolation, identifications and tissue localization in the tiger prawn Penaeus monodon. Eur. J. Biochem. 267,1153 -1160.[Medline]
Kubiak, T. M., Larsen, M. J., Burton, K. J., Bannow, C. A., Martin, R. A., Zantello, M. R. and Lowery, D. E. (2002). Cloning and functional expression of the first Drosophila melanogaster sulfakinin receptor. Biochem. Biophys. Res. Commun. 291,313 -320.[CrossRef][Medline]
Kuramoto, T. and Ebara, A. (1984). Effects of
perfusion pressure on the isolated heart of the lobster Panulirus
japonicus. J. Exp. Biol.
109,121
-140.
Kuramoto, T. and Ebara, A. (1985). Effects of perfusion pressure on the bursting neurons in the intact or segmented cardiac ganglion of the lobster, Panulirus japonicus. J. Neurosci. Res. 13,569 -580.[CrossRef][Medline]
Maestro, J. L., Aguilar, R., Pascual, N., Valero, M. L., Piulachs, M. D., Andreu, D., Navarro, I. and Belles, X. (2001). Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins? Eur. J. Biochem. 268,5824 -5830.[Medline]
Mercier, A. J. and Russenes, R. T. (1992). Modulation of crayfish hearts by FMRFamide-related peptides. Biol. Bull. 182,333 -340.[Abstract]
Monigatti, F., Gasteiger, E., Bairoch, A. and Jung, E.
(2002). The Sulfinator: predicting tyrosine sulfation sites in
protein sequences. Bioinformatics
18,769
-770.
Nachman, R. J., Holman, G. M., Haddon, W. F. and Ling, N.
(1986a). Leucosulfakinin, a sulfated insect neuropeptide with
homology to gastrin and cholecystokinin. Science
234, 71-73.
Nachman, R. J., Holman, G. M., Cook, B. J., Haddon, W. F. and Ling, N. (1986b). Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin. Biochem. Biophys. Res. Commun. 140,357 -364.[CrossRef][Medline]
Nachman, R. J., Giard, W., Favrel, P., Suresh, Y., Sreekumar, S.
and Holman, G. M. (1997). Insect myosuppressins and
sulfakinins stimulate release of the digestive enzyme -amylase in two
invertebrates: the scallop Pecten maximus and insect
Rhynchophorus ferrugineus. Ann. N. Y. Acad. Sci.
814,335
-338.[CrossRef]
Nachman, R. J., Vercammen, T., Williams, H., Kaczmarek, K., Zabrocki, J. and Schoofs, L. (2005). Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects. Peptides 26,115 -120.[CrossRef][Medline]
Nichols, R. (1992). Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol. Cell. Neurosci. 3,342 -347.
Nichols, R. and Lim, I. A. (1996). Spatial and temporal immunocytochemical analysis of drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system. Cell Tissue Res. 283,107 -116.[CrossRef][Medline]
Nichols, R., Schneuwly, S. A. and Dixon, J. E.
(1988). Identification and characterization of a
Drosophila homologue to the vertebrate neuropeptide
cholecystokinin. J. Biol. Chem.
263,12167
-12170.
Passano, L. M. (1951). The X-organ-sinus gland system in crabs. Anat. Rec. 111, 502.
Predel, R., Brandt, W., Kellner, R., Rapus, J., Nachman, R. J. and Gäde, G. (1999). Post-translational modifications of insect sulfakinins. Eur. J. Biochem. 263,552 -560.[Medline]
Schoofs, L., Holman, G. M., Hayes, T. and De Loof, A. (1990). Isolation and identification of a sulfakinin-like peptide with sequence homology to vertebrate gastrin and cholecystokinin, from the brain of Locusta migratoria. In Chromatography and Isolation of Insect Hormones and Pheromones (ed. A. R. McCaffery and I. D. Wilson), pp. 231-241. New York: Plenum Press.
Selverston, A. I. and Moulins, M. (ed.) (1987). The Crustacean Stomatogastric System. Berlin: Springer.
Torfs, P., Baggerman, G., Meeusen, T., Nieto, J., Nachman, R. J., Calderon, J., De Loof, A. and Schoofs, L. (2002). Isolation, identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropod, the white shrimp Litopenaeus vannamei. Biochem. Biophys. Res. Commun. 299,312 -320.[CrossRef][Medline]
Towle, D. W. and Smith, C. M. (2006). Gene
discovery in Carcinus maenas and Homarus americanus via
expressed sequence tags. Integr. Comp. Biol.
46,912
-918.
Turrigiano, G. G. and Selverston, A. I. (1989). Cholecystokinin-like peptide is a modulator of a crustacean central pattern generator. J. Neurosci. 9,2486 -2501.[Abstract]
Turrigiano, G. G. and Selverston, A. I. (1990). A cholecystokinin-like hormone activates a feeding-related neural circuit in lobster. Nature 344,866 -868.[CrossRef][Medline]
Turrigiano, G. G., Van Wormhoudt, A., Ogden, L. and Selverston, A. I. (1994). Partial purification, tissue distribution and modulatory activity of a crustacean cholecystokinin-like peptide. J. Exp. Biol. 187,181 -200.[Abstract]
Veenstra, J. A. (1989). Isolation and structure of two gastrin/CCK-like neuropeptides from the American cockroach homologous to the leucosulfakinins. Neuropeptides 14,145 -149.[CrossRef][Medline]
Veenstra, J. A. (2000). Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine peptide precursors. Arch. Insect Biochem. Physiol. 43, 49-63.[CrossRef][Medline]
Wei, Z., Baggerman, G., Nachman, R. J., Goldsworthy, G., Verhaert, P., De Loof, A. and Schoofs, L. (2000). Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria. J. Insect Physiol. 46,1259 -1265.[CrossRef][Medline]
Worden, M. K., Clark, C. M., Conaway, M. and Qadri, S. A.
(2006). Temperature dependence of cardiac performance in the
lobster Homarus americanus. J. Exp. Biol.
209,1024
-1034.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
