Characterization of Two Novel Neuropeptides From the Sea Cucumber Holothuria glaberrima

7 Pages·1992·2.7 MB·English

by  L Diaz-Miranda

Reference: Biol. Bull 182: 241-247. (April. 1992)
Characterization of Two Novel Neuropeptides From
the Sea Cucumber Holothuria glaberrima
TLEURCRYYDID.AZL-EMEI3RAKNADRAE1N. DEA.VDIODBLA.E2PRAINCED2.JMOISECHEA.EGLARJ.CGIAR-EAERNRBAERRAGS21,
, ,
^Department ofBiology, University ofPuerto Rico, Rio Piedras, Puerto Rico 0093J; 2The Whitney
Laboratory, St. Augustine, Florida 32086; and ^Division ofImmunology, Beckman Research
Institute ofthe CityofHope, Duarte, California 91010
Abstract. Two peptides were purified from intestinal immunoreactive nerve fibers were found in the area of
extracts of a sea cucumber, Holothuria glaberrima, by the tube feet, suggesting that FMRFamide might be reg-
high pressure liquid chromatography (HPLC). The pep- ulating the process oflocomotion (Elphick el al, 1989).
tides were detected by a radioimmunoassay (RIA) based Subsequently, two novel neuropeptides from the starfish
on an antiserum raised to the molluscan peptide, pGlu- A. rubens and A. forbesi were identified: GFNSALM-
Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide). Famideand SGPYSFNSGLTFamide, andthepreviously
Automated sequencing and mass spectrometry indicate reported FMRFamide-like immunoreactivity inA. rubens
that the isolated peptides are: Gly-Phe-Ser-Lys-Leu-Tyr- was attributed to these peptides (Elphick et al.. 1991).
Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val- Peptide immunoreactivity has also been demonstrated
Leu-Tyr-Phe-NH2(SGYSVLYFamide). Thesearethefirst in members ofanother echinoderm class, the Holothu-
peptidestohavebeenisolated from a memberoftheechi- roidea. For example, cholecystokinin (CCK)-like immu-
noderm class Holothuroidea. noreactivity occurs in neurons and in a plexus of fibers
Theantiserum used inthe RIA ofthe peptideswasalso in the intestinesofHolothuria mexicana, Holothuriagla-
employed in localizing immunoreactive nerve cells and berrima, andStichopusbadionotus(Garcia-Arrarasetal.,
fibers in the intestine of//, glaberrima. The immunohis- 199la). Similarly, FMRFamide-like immunoreactivity
tochemical results suggest that these peptides might be was reported in cells and fibers ofthe intestine ofH. gla-
responsible for the FMRFamide-like immunoreactivity berrima (Garcia-Arraras et al., 1991b). The location of
reported earlier. Sequence similarities between these immunoreactive substances suggeststhat they have
GFSKLYFamide, SGYSVLYFamide, and a pair ofpep- arolein the regulation ofdigestive physiology, and indeed
tides previously isolated from starfish lead us to propose peptidesoftheCCK familydo induce a partial relaxation
thatall fourmoleculesaremembersofafamilyofpeptides of the intestinal musculature (Garcia-Arraras et al..
acting as neurotransmitters in echinoderms. 199la). These results notwithstanding, none of the en-
dogenouspeptidesin the nervoussystem ofseacucumbers
Introduction had been identified before the present study was under-
taken.
Very few echinoderm neuropeptides have been char- In this report, we describe the isolation and character-
acterized. For example, the sequence ofthe first neuro- ization oftwo peptides from the digestive system of H.
peptide detected in this phylum i.e., gonad-stimulating glaberrima. In addition, we provide histochemical evi-
substance(GSS) from starfish(Chaetand McConnaughy,
dence for the presence of these peptides in the enteric
1959) is still unknown (references in Cobb, 1988). Re- nervous system ofthe sea cucumber.
cently, FMRFamide-like immunoreactivity was detected
in the nervous system ofthe starfish Asterias rubens, and Materials and Methods
Specimensof//, glaberrima(10-15 cm in length)were
Received 10October 1991;accepted 27January 1992. collectedfrom therocky intertidalzoneofthenorthcoast
241
242 L. DIAZ-MIRANDA ET AL
ofPuerto Rico. Theanimalswereeitherused immediately halfwas dried in a Speed-Vac for FABms, and the other
or, in some cases, maintained in marine aquaria at the half(about .1 ml) was applied (in 3 portions with inter-
Department ofBiology ofthe University ofPuerto Rico. mediate drying) directly to a pre-conditioned glass-fiber
filter disk containing 3 mg of Polybrene. The disk was
Digestivesystem extracts placed in the sequencer (Applied Biosystems 470A gas-
Four extracts ofsea cucumber digestive systems were tphheasPeTsHe-qaumenicneorwaicitdhdaenriovna-tliivnees1i2n0eaaPchTHcycalnealwyezerre),idaennd-
prepared as follows. First, 13 to 25 animalswere sectioned tified by their retention times and quantitated by com-
with a razor blade, just posterior to the calcareous ring. parison ofthe peak areas to standards (performed by B.
The body wall was slit open, and the intestinal tract, in-
cluding the esophagus and the small and large intestine, BPairotteenc,hnUonlivoegrysiRteyseoafrcFhl,orPirdoatIenitnerSdeiqsuceipnlciinnagryCCoerneteFarciflo-r
troatgoertyhertrweei,thweardheerreenmtovpeidec.esTohfehetimsaslueve(s3s7el-1a1n3dgreswpeit- aityJ,EGaOiLnesHvXil1le0)0.HTFhemFagAnBemtiscasneacltyosrismwaassscsaprerciterdoomuetteorn,
weight)wasplaced ina fourfold excessofacetoneand left
at 20C for 48 h. The supernatant was then filtered as described in Bulloch et al. (1988).
through Whatman #1 paper, and the acetone and part of
the water were removed on a rotaryMevaporator. The Syntheticpeptide
aqueous portion was acidified to 0.02 acetic acid, cen- The peptides GFSKLYFamide and SGYSVLYFamide
trifugedat 2500 X g. andthesupernatantdriedinaSpeed- were synthesized on an Applied Biosystems synthesizer
Vac (Savant). The dried sample was reconstituted in bythe Protein Chemistry Laboratory ofthe University of
aqueous0.1%trifluoroaceticacid(TFA),centrifuged, and Florida's Interdisciplinary Center for Biotechnology Re-
filtered. search; t-Boc protecting groups were used. The peptides
were deprotected and removed from the resin with tri-
Purification fluoromethanesulfonic acid (Applied Biosystems proto-
Thereconstituted samplewaspumped ontoa Brownlee col), purified by HPLC, and quantified by amino acid
C8 reverse phase column (Aquapore RP300: 220 X 4.6 analysis (Hitachi 835 analyzer).
mmorPrep 10AquaporeOctyl 100 X 10mm), according
Immunohistochemistry
to the procedure described by Price el al. (1990a). Once
loaded, thecolumn wasrinsed withaqueoussolvent(0.1% For the histochemical study, the procedure described
TFA) until the UV absorbance fell to near baseline. The by Garcia-Arraras et al. (199la) was followed. In brief,
eluting solvent was rapidly changed (by a step or 1 min isolated portions ofthe large and small intestines ofH.
gradient)to 20 or 30% oforganicsolvent (80% acetonitrile glaberrimawerefixedinpicricacid-formaldehyde mixture
containing 0.1% TFA), whereupon a linear gradient was overnight at 4C. The tissue sections (10-12 ^m) were
started with a 1%/min increase in the organic solvent up treated with antiserum Q2 (1:500) or with an antiserum
to 50 or 60% organic. Half-minute fractions were col- against FMRFamide (#8 3i 2s; 1:500) (Garcia-Arraras et
lected, and 2 n\ aliquots were taken from each fraction a/., 1991b). Asacontrol, theQ2 antiserum wasincubated
for the RIA. with 10 Mg/ml ofGFSKLYFamide, FMRFamide (Pen-
Furtherpurification wasalsodoneon BrownleeC8 RP- insula), or FLRFamide (Sigma) for 24 h before being ap-
300 columns (220 X 2.1 mm or 220 X 4.6 mm). The plied to the tissue sections.
columns were developed, either with TFA/acetonitrile
gradients as above, or with aqueous 0.05% heptafluoro- Results
butyric acid (HFBA) and 80% acetonitrile containing
0.05% HFBA. Fractionation ojextracts
Each ofthe four gut extracts was fractionated with a
Radioimmunoassay (RIA) somewhat different series ofHPLC steps. We discovered,
wFaLsARFadrimalibubdtieetdcoa1un:pt5li0es0dertufomotrhy(ruQos2ge)l,obinurlaiitnshe(edPriRacIgeaAei.tnsalt.l.odp1i9Qn9Da0tbPe)-d, epfieanpcathlilysd,teestph,wataanstdhteotsosiermlupenlcettstthhewemaiybmamtcouknpuotrhrirefoayuctgthhievtiehmemfsruaancmoterieocnaoscltaufitmvener
pQYPFLRFamide served as the tracer. under the same conditions (Fig. 1). This finding was cer-
tainly notexpected. The methodworksbecausetheextract
Sequencing andspectrometry behavesanomalously; i.e., theimmunoreactive peaksshift
toan everearlierelution timeduringeach successive step
The most immunoreactive fraction within each pure ofpurification (e.g.. compare Figs. 1C2 and 1C3). More-
peak wasanalyzed. The fraction wasdivided in half: one over, even the order of elution of the immunoreactive
NOVEL NEUROPEPTIDES FROM SEA CUCUMBERS 243
10 20
B4 100-1 r2 Elution Time (min)
10 20
Elution Time (min)
244 L. DIAZ-MIRANDA ET AL
peaks changes (Fig. 1). This relative shift in elution po-
sition was clearly observed in all but the first ofthe four
extracts.
One peptide,alreadypurified fromthe firstextract,was
readilyandunambiguously identifiedasGFSKLYFamide
when weobtaineditsmolecularion (860.4)and sequence
(Fig. 2a). The calculated value of the amide of this se-
quence is 860.5, whereas that of the free acid is 861.5.
Thus the molecular ion confirmed the presence ofthe C-
terminal amide, which had been inferred from the im-
munoreactivity; in contrast, the PTH derivatives ofphe-
nylalanine and its amide are indistinguishable in normal
Edman sequencing. The first extract contained other mi-
norimmunoreactive peaks; one ofthese wasanalyzed by
FABms and sequenced; this product, found again in the
fourth extract, will be described further below.
From the second extract, we obtained a molecular ion
(934.6) and the partial sequence of a second peptide,
SGXSVLXFamide (where X could be either tyrosine or
methioninesulfone). Thefirstpeptide(GFSKLYFamide)
did not appear in this extract.
Thethirdextractcontained two immunoreactive peaks.
Using mass spectrometry, we again identified GFSKLY-
Famide(860 molecularion)and SGXSVLXFamide(934
molecular ion).
The fractionation ofthe fourth extractwas undertaken
toresolvetheambiguities left afterthe first three, and the
HPLC runs leading to the purification of the two most
immunoreactive peaks are shown in Figure 1. 456
The first HPLC step in this last purification yielded a 10
broad, irregularpeakofimmunoreactivity(Fig. 1A). From Cycle Number
the earlier halfofthis, we succeeded, after four steps, in Figure 2. Aminoacidsequencesofthepurifiedpeptides.Theyields
purifying a peak (at 15 min in Fig. 1B4) that sequenced ofthe pertinent PTH amino acid derivatives at each cycle are plotted
as SGYSVLYF (Fig. 2B). The calculated molecular ion andtheaminoacidassignedtoeach position isshown.(A)Thepeptide
forthispeptide with itsC-terminal amidated is934.5, havingan 860 molecular ion. (B) The peptide havinga934 molecular
ion.
and this is in good agreement with the molecular ion
(934.6) found earlier. The small immunoreactive peak at
16 min in Figure 1B4 had the same sequenceasthe main
peak, so it is probablyjust a tail ofthe main peak. afterSGYSVLYFamide(seearrowsin Figs. 1B1 and 1C1).
From thelaterhalfoftheinitial broad immunoreactive Afterpurification (not shown), these pooled minor peaks
peak obtained in the first step of the purification (Fig. yielded the sequence GFSXLYF, which corresponds to
1A), two peaks were resolved in the second (Fig. 1C1). that of the synthetic peptide, except that no lysine (or
The major (and earlier-eluting) ofthese peaks co-eluted otherPTH derivative) appeared in cycle4. This peak had
with synthetic GFSKLYFamide. a molecular ion of958, which is 98 larger than that ex-
The second peak, eluting at 23.5-25 min, was pooled pectedforthelysinecontainingamidated peptide. Apeak
with a similar small peak that had eluted a few minutes with the same relative elution time, and a similar molec-
Figure 1. HPLC fractionation ofa sea cucumber gut extract. The ultraviolet absorbance at 210 nm
(solid line) and the immunoreactivity (histogram)areshown foreach HPLC run. The initial fractionation
(A)wasdoneonaPreplOOctylcolumn(10 x 100 mm;4 ml/mm)witha30 mingradientfrom 16to40%
acetonitrileinwaterwith0.1%trifluoroaceticacid. Subsequent runs(B1-4:Cl-3)weredoneon an RP-300
column(2.1 > 220 mm;0.5 ml/min)with thesamegradient. Thearrowsin Bl andCl indicatepeaksthat
were pooled and subsequently purified (not shown; see text). The full scale absorbance in the top trace is
2.0and is0.5 AU in all thesubsequent traces.
NOVEL NEUROPEPTIDES FROM SEA CUCUMBERS 245
ular ion (958.6) and sequence, had also appeared in the
first extract, as briefly noted above.
We suspect that the peptide GFSXLYFamide has a
derivatized lysinesidechain. Forexample, a peptide with
the lysineaminogroupamidated by hexanoicacid would
give such a molecular ion. and such a derivative could
occur naturally in the sea cucumber. It is more likely,
however, that the derivative is a substituted oxazolidine
ring, formed by the condensation, with the lysine, oftwo
molecules ofacetone (58 + 58) with the loss ofa water
(18). Thisderivatization would add 98 to the molecular
weight ofthepeptide. In summary, this peptide may very
well be an artifact ofthe purification.
Syntheticpeptides
The synthetic peptidesGFSKLYFamide and SGYSV-
LYFamide were purified by HPLC after deprotection.
Each peptide co-eluted with its natural counterpart on
HPLC. An amino acid analysis ofGFSKLYFamide gave
the following composition: Gly 1.05, Phe 1.75, Ser 1.0,
Lys 1.1, Leu 1.25, and Tyr 1.05. The composition of
SGYSVLYFamide was: Gly 1.00, Phe .90, Ser 2.03, Leu
1.11, Tyr 1.99, Val 1.33.
Figure3. Transversesectionsofthelargeintestineof//,glaberrima
Q2 immunoreactivity showingQ2-like immunoreactivity. A. Immunoreactive nerve fibersat
theleveloftheserosaandlongitudinal muscle. Mostofthesefiberswere
Thetissuedistribution ofimmunoreactivity toQ2 (the associatedwiththelongitudinalmuscle(arrowhead).B.Arrowheadpoints
antiserum used in the characterization ofGFSKLYFam- atoneimmunoreactivefiberextendingthroughoutthesubmucosalayer.
Asterisk: endogenous fluorescence. Magnification: A. X405; B. X270.
ide and SGYSVLYFamide) was determined by immu-
nohistochemistry. ResultswithantibodiesQ2and #8(the
latter an antiserum against FMRFamide) were similar. was not blocked, whetherthe antiserum was preabsorbed
Cellsand fibers located in the outerconnective tissue (se- with FMRFamide or with FLRFamide.
rosa)ofthesmall and large intestinewerelabeled, aswere
single fiber-like projections in the submucosal layer (Fig. Discussion
3). In addition, a strong nerve plexuswas observed in the
mesentery next to the muscular layer. This nerve plexus We have purified two peptides from gut extractsofthe
wascontinuous with the serosal nerve plexus. In contrast sea cucumber H. glaberrima, using high pressure liquid
tothe FMRFamide-like immunoreactivity (Garcia-Arra- chromatography for separation and radioimmunoassay
ras et ai, 1991b), Q2 also labeled a group ofcells located for detection. These peptides GFSKLYFamide and
in the submucosal layer ofthe intestines, similar to what SGYSVLYFamide were completely characterized by
has been described as morula cells (Hetzel, 1963, 1965), microsequencingand massspectrometry,andarethefirst
but this reaction does not seem to be specific. to have been isolated from the echinoderm class Holo-
When the Q2 antiserum was preabsorbed with thuroidea.
GFSKLYFamide (12 jiM), no immunoreactivity to Q2 Tworelated peptideswereisolatedearlierfromanother
was observed in the cells and fibers ofthe mesentery, se- echinoderm class, the Asteroidea: i.e.. GFNSALMFamide
rosa, or submucosa; but the morula cellscontinued to be and SGPYSFNSGLTFamide from the starfishesAsierias
labeled. These results suggest that otherantibodies in the forbesiandA. ntbens(TableI; Elphicketal., 1991). These
Q2 serum recognize unrelated substances. The peptides peptides, like those of the sea cucumber reported here,
FMRFamide (17 fiM)and FLRFamide(17 pM)werealso weredetectedon thebasisoftheirbindingtoanantiserum,
used for preabsorption ofQ2, but they could not com- Q2, raised against pQDPFLRFamide (Price et a/.. 1990b).
pletely block the observed immunoreactivity ofthe cells In both studies, furthermore, the Q2 antiserum was orig-
and nerve fibers in the serosa, orofthe nerve fibers in the inallyselectedbecausetheaimwastocharacterizeputative
submucosa. The Q2 immunoreactivity ofthe morula cells FMRFamide-related peptides (FaRPs) that had been
246 L. DIAZ-MIRANDA ET AL.
Table I
AminoacidsequencesofSxLxFamide1 peptidesisolatedfromEchinodermata
CLASS
Species Sequence Ref.
HOLOTHUROIDEA
Holothuriaglaberrima Gly-Phe-SER-Lys-LEU-Tyr-PHE-NH2
Ser-Gly-Tyr-SER-Val-LEU-Tyr-PHE-NH2
ASTEROIDEA
Asteriasforbesi Gly-Phe-Asn-SER-Ala-LEU-Met-PHE-NH2
A. rubens Ser-Gly-Pro-Tyr-Ser-Phe-Asn-SER-Gly-LEU-Thr-PHE-NH2
1 Ser-x-Leu-x-Phe-NH:.wherethepositions"x"canbeoccupiedbyany residue.
Thisreport.
3Elphickei al.. 1991.
identifiedbyimmunocytochemistry (Elphickelal, 1989; munoreactivity previously reported in both echinoderm
Garcia-Arraras et a/., 1991b). classesmightbeduetothepresenceoftheisolatedpeptides
FaRPs, defined liberally, have now been isolated from reacting with the anti-FMRFamide serum.
many animal groups, including coelenterates, nematodes, As Table I illustrates, the two sea cucumber peptides
annelids, arthropods, and even vertebrates (reviewed by have five ofseven residues in common, and the two star-
Price and Greenberg, 1989; Greenberg and Price, 1992), fish peptides have five of eight identical; but the most
and a penultimate arginyl residue is not only a common similarsea cucumberand starfish peptidesshare only four
feature ofthisextended family, but has been shown to be ofeight residues. The clear unifying feature ofthese four
critical for physiological activity (e.g., see Kobayashi and echinoderm peptides is the C-terminal sequence SxLx-
Muneoka, 1986). Antiserum Q2 should have identified Famide, where the positions indicated by "x" can be oc-
most FaRPsthat might havebeen present in ourextracts, cupied by any other residue. We therefore propose that
but in fact, not one ofthe four neuropeptides sequenced this periodic sequence ofserine, leucine, and phenylala-
from echinoderms has the penultimate arginine. Our re- nine defines a novel family of peptides present in the
sults, therefore, when taken together with the evidence Echinodermata.
obtained from the starfish (Elphick et al., 1991), suggest
that authentic FaRPsareabsent from the Echinodermata.
Acknowledgments
Ifthe above assertion is correct, we must account for
the FMRFamide-like immunoreactivity described in sea This research was supported by funds from the Re-
cucumbers (Garcia-Arraras el al., 199Ib) and in starfish source Center for Sciences and Engineering, the Patricia
(Elphickelal.. 1989). IntheseacucumberH.glaberrima, Robert Harris Fellowshipto LDM, theGrassFoundation
immunoreactivity to FMRFamide has been reported in (to the Whitney Lab), the National Science Foundation
the radial nerves, in nerve plexuses ofthe esophagus, and (BNS-8801538 to JGA), and the National Institute of
in the enteric nervous system (Garcia-Arraras et al., Health (HL28440 to MJG). We would like to thank the
1991b). The distribution of immunoreactivity observed Protein Chemistry Core Facility of the University of
withtheQ2antibodyinthegutof//. glaberrimaissimilar, Florida forpeptidesequencingandpeptidesynthesis, and
ifnot identical, to that observed with antibodies against we also acknowledge the help of the Interdisciplinary
FMRFamide. The localization of Q2-binding to nerve Center for Biotechnology Research (ICBR) of the Uni-
cellsand fibersin theseacucumberintestinesuggeststhat versity of Florida. We are very grateful to Mr. Dietmar
the peptides recognized by this antibody occur in the en- Nieves and Ms. Lynn Milstead for their excellent assis-
teric nervous system ofholothurians, and that they may tance with the preparation offigures.
be involved in neural transmission or modulation.
The story in the starfish Asterias rubens is similar: i.e., Literature Cited
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e1p9i8t9h)e,liaalndnetrhivseipmlmeuxnuosreoafctthievittuybehasfeebtee(nElapthtircikbuettedalt.o, pBhoywseiso.lo1g9i8c8a.l acFtiMoRnFsaamtiadpeerpiepphteirdaelssiynnHaepslei.so/ma:Neiudreonstciif.ica8t:io3n45a9n-d
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