THEJOURNAL OF BIOL~CICAL CHEMISTRY Vol. 269, No. 28, Issue of July 15, pp. 18295-18298, 1994 0 1994 by T h e American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication
crayfish, Orconectes limosus (3) and Procambarusbouvieri (4), a n d lobster, Homarus americanus (5, 6). In the latter species, the complete sequences of the two variants (cHHAand cHHB) were established by polymerase chain reaction (7) and showed 92% homology, with the first 19 amino acids being identical (Table I). In addition,it has been observed consistentlythat t h e D-AMINOACID RESIDUE IN CRUSTACEAN as doublet peaks. The two cHHs elute from HPLC columns HYPERGLYCEMIC PEPTIDES* components of cHHA and cHHB were purified to homogeneity, (Received for publication, January 5, 1994, and in revised form, and it was demonstrated that they had the same molecular April 6, 1994) mass, pHi, and amino acid composition (8)a n d that their amino at least up to the 20th residue (9). acid sequence was identical, Daniel SoyezSS, Franpois Van Herpll, The molecular masses measured by mass spectrometric techJean RossierlI, Jean-PierreLe Caerli, Cees P. Tensenll, and Ren6 Lafont$ niques are identical with those calculated from the amino acid of t h e N- and sequence, showing that, except for modifications From the $Laboratoire de Biochimie et Physiologie du C-terminal groups, no other obvious post-translationalmodifiDeueloppement, URA CNRS 686, Ecole Normale Superieure, 46 rue d'Ulm, 75230 Paris cedex, France, cations (e.g. glycosylation or sulfatation) take place. All these the IDepartment of Experimental Zoology, Faculty of data suggested that cHHA and cHHB are the products of two Sciences, Catholic University of Nijmegen, Toernooiveld, different genes, but they did not explain the existence of two NL6525 ED Nijmegen, The Netherlands, a n d the isoforms for each peptide. lpnstitut Alfred Fessard, CNRS, The present study was initiated in order to elucidate the 91198 Gif sur Yvette, France structural basis of t h e difference between the cHHs isoforms. Several large peptidic neurohormones have been iso- We initially showed that t h e isoforms are present, witha conlated in crustaceans. In lobster and other related spe- stant ratio, in a single sinus gland. Then we demonstrated, cies, each of these neurohormones, and particularly the usingproteolyticdigestion,mass spectroscopy, andchiral crustacean hyperglycemic hormone, occursas two iso- amino acid analysis, that the cHHs isoforms differ by the conforms having the same peptidic sequence and molecular 3 of their formation of the phenylalanyl residue in position mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third sequence. Finally, the physiological signification of this polyis morphism was studied by determining the time course of t h e residue (Phe3)of the lobster hyperglycemic hormones of t h e differin either the L- or D-configuration. In addition, we have hyperglycemic response in crayfish after injection ent cHH isoforms. shown that the biological activity of the two isoforms
Evidence for a Conformational Polymorphism of Invertebrate Neurohormones
differs when considering the kinetics of their hyperglycemic effect.
MATERIALS AND METHODS Purification ofHyperglycemicPeptides-Hyperglycemic peptides were extracted from sinus glands of 300-500-g American lobsters (H. umericanus). Physiological status of the donors (sex, molting, and reproductive stage) was not recorded. Eyestalks were stored lyophilized In decapod crustaceans, molting, reproduction, or glycemia and were rehydrated just before the dissection of the sinus gland. Sinus are controlledbyneurohormonesoriginatingfromtheeyestalks. These hormones are synthesized by paired clusters of gland peptides were extracted with 10% acetic acid at 80 "C for 5 min and purified by a single RP-HPLC step as described previously (11). neurosecretory cells (called X-organs),then stored in neuroheMicro-HPLCAnalysis oflndividual SinusGlands-Separation of the mal organs (the sinus glands) and later released into the he- four hyperglycemic peptides from individual sinus glands was carried molymph. The most abundant neurohormone stored in sinus out with the SMARTTMsystem (Pharmacia LKB, Uppsala, Sweden). glands is the crustacean hyperglycemic hormone (cHH)' in- The left and right sinus glands were collected from living anesthesized volved in animal homeostasis and stress response,via the regu- lobsters and immediately frozen in dry ice-cooled glass-glass microholation of glycemia through the classical mechanisms of glyco- mogenizers. Afterward, the sinus gland samples were homogenized in approximately 350 pl of 0.1 M HCl, heated at 80 "C for 5 min, and then gen mobilization. The primary structure of the cHH froma few freeze-dried. The dried samples were dissolved in 45 p1 of 0.1% trifluspecies has been recently established, demonstrating that all oracetic acid. Aftercentrifugation at 12,000 x g for 30 min, the superthese neuropeptides contain 71 or 72 amino acid residues, have natant wasinjectedonto the SMART system. Separation was pera high degreeof homology (>60%),a n d share common features formed on a reversed phase precision column: type pRPCC2/C18,2.1 x 100 mm (SC 2.1/10), 3-pm particle size, and 350-pl gel volume. The such as 6 fully conserved cysteine residues. They constitute, peptides were eluted by a linear gradient of solvent B (80%acetonitrile, together with other eyestalk neurohormones, a new peptide 20%water, 0.1% trifluoracetic acid) in solventA(O.l%trifluoracetic acid family (for a review, see Ref. 1). in water), with a flow rate of 200 pllmin. The W absorbance was The presence of molecular variants of the cHH froma given recorded at 214 nm using the W-MI1pPeak Monitor (Pharmacia LKB) species has been reported in crab, Cardisoma carniflex (2), and the chromatographic data from individual sinus glands evaluated with the SMART Manager Software. Peptide Fragmentation and Purification of Fragments-Hypergly* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked cemic peptides (approximately 1 nmol) were fragmented by endoproteinase Lys-C (sequencing grade, Boehringer Mannheim) according to uadvertisernentnin accordance with 18 U.S.C.Section1734solelyto Ref. 9. Purified peptides weredigested by 0.5 pg of endoproteinase indicate this fact. 0 To whom correspondence should be addressed. Tel.: 33-1-44-32-36- Lys-C in 250 pl of digestion buffer(25mM Tris-HC1, pH 8, 1 mM EDTA, and 5 mM dithiothreitol) for 16 h at 20 "C. The digestion mixture was 28; Fax: 33-1-44-32-39-10;E-mail:
[email protected]. ' The abbreviations used are: cHH, crustacean hyperglycemic hor- injected onto the HPLC column (4.6 x 250 mm packed with Nucleosil mone; HPLC, high performance liquid chromatography; RP-HPLC, re- C-18, 5-pm pore size) after dilution with 750 pl of water, 0.1%trifluversed phase HPLC; FDNPA, l-fluoro-2,4-dinitrophenyl-5-~-alanine. oracetic acid, and the fragments were eluted with a discontinuous gra-
18295
D-Amino Invertebrate Residue Acid in
18296
Neurohormones
TABLEI Primary structure of the two lobster hyperglycemic hormones, according to Ref. 7 The aminoacid residues that differ between the two hormones are underlined. The N-terminal octapeptide obtained by enzymatic cleavage (as described in Fig. 2) is boxed. 5
10
15
20
25
30
35
CHH A
I
pGlu-Val-Phe-AspCln-Ala-Cys-Lys~ly-Val-Tyr-Asp~g-Asn-Leu-~e-Lys-Lys-Leu-Asp~g-Val~ys.Glu-AspCys-Tyr-Asn.Leu-Ty~-Arg-Lys-Pm-Phe-Val-Ala...
CHH B
I
pGlu-Val-Phe-AspGln-Ala-Cys-Lys~ly-Val-Tyr-Asp~g-A~n-Leu-~e-Lys-Lys-Leu-Asn-Arg-Val-Cys-Glu-AspCys-Tyr-Asn-Leu-Tyr-ArgLys-Pro-PheIle- Val-
-
-
...
40 45 50 55 60 65 70 Thr-Thr-Cys-Arg-Glu-AsnCys-Tyr-Ser-Asn-~Val-Phe-Arg-Gln-Cys-Leu-Asp-Asp~u~Leu-~Asn-ValIle- Asp-Glu-Tyr-Val-Ser-Asn-Val-Gln-MelVal h ~
...
Thr-Thr-Cys-Arg-Glu-Asn-Cys-Tyr-Ser-Asn-Arg-Val-Phe-Arg-Gln-Cys-Leu-AspAsp~u-Leu-Ile-Asn-ValIle- Asp-Glu-Tyr-Val-Ser-Asn-Val-Gln-MelVal MI?
2
dient of acetonitrile in water, both solvents containing 0.1% trifluoroaceticacid.Thepeakswere collected, aliquoted,andevaporated t o dryness in a Speed-Vac centrifuge concentratorfor further analysis. Mass Spectroscopy-Fragments were dissolved in 30 plof a mixture of acetonitrile/water/formic acid (50/49/1). Sample solution (10 pl) was injected ontothe electrospray sourceof a VG Trio 2000 quadrupole mass spectrometer via a loop injector at a flow rate of 3 pllmin and was scanned over a mass to charge ratio (m/z) between 400 and 1200. The instrument was calibrated withmyoglobin (molecular mass = 16,951.5 Da). Amino Acid Analysis-Chirality of amino acid residues was determined by the method describedby Scaloni et al. (10). The chiral reagent l-fluoro-2,4-dinitrophenyl-5-~-alanine (FDNPA) used for pre-column derivatization was synthesized in the laboratoryfollowing the procedure of the aforementioned authors. Peptide fragments werehydrolyzed with 100 plof 6 M constant boiling hydrochloric acid (Sigma) in vacuum-sealed ampules for 16 h at 110 "C. After hydrolysis, the acid was evaporated in a Speed-Vac concentrator and the dry residue dissolved in 50 pl of ultrapure Milli-Q water. The aminoacid solution was transferred intoa small Eppendorf tube and evaporated to dryness again. Fifteen microlitersof 0.2 N sodium bicarbonate were added. After shaking,15 p1 of FDNPA solution FIG.1. Microchromatography of an extract from a single lobin acetone (containing approximately 2 nmol) were added to the tubes. crustacean hyperster sinus gland showing the isoforms of the Coupling was realized by incubation under gentle shaking for 1 h at glycemichormonesand of the vitellogenesis inhibiting hor50 "C in the dark. mone (VZH).These peptides were identified on the chromatogram by Separation of DNPA derivatives was carried out on Nucleosil a C-18 comparison with previously published analysis of batch sinus gland (5 pm particle size) column (4.6x 250 mm). Elution was realizedby a samples (5, 6). Chromatographic conditions were as follows. Column gradient of acetonitrile/2-propanol (4/1) in triethylamine/phosphoric length was100 mm, witha 2.1-mm internal diameter.Stationary phase acid buffer (40mM, pH 2.2)a t a flow rate of 1 mumin. UV absorbance at was pRPC C2/C18 (3-pm particle size). Solvent A was 0.1% trifluorace340 nm was monitored by a LDC 2000 spectrophotometer. Identification tic acid in water,solvent B was 80% acetonitrile, 20% water, 0.1% of the derivatives was madeby comparison with similar analysis of Ltrifluoracetic acid. Flow rate was 200 pllmin. The elution gradient is shown by the dotted line. and D-amino acid standard solutions (not shown). Synthetic Peptides-Peptides pGlu-Val-Phe-Asp-Gln-Ala-Cys-Lys and pGlu-Val-o-Phe-Asp-Gln-Ala-Cys-Lys were synthesizedby Neosyschromatographic method. We have demonstrated that the four tem Lab (Strasbourg, France). After reduction of the cysteine residueby hyperglycemic peptides are present in a singlesinus gland (Fig. dithiothreitol, the peptides were further purifiedby RP-HPLC. found in sinus glands from males and Bioassays-Forty pmol of cHHA or [ D - P ~ ~ ~ I c Hdissolved HA, in physi- 1). The same pattern was ological saline, were injected into crayfish (0.limosus) at the beginning females. Moreover, a quantitative analysis of left and right of the experiment. Aliquots (25 p1)of hemolymph were withdrawn a t sinus glands from 5 lobsters showed that the relative abundifferent time intervals. Hemolymph glucose levels were determined, dance of isoforms was constant. The ratio of the more polar after precipitation of the proteins with perchloric acid, using the Pericomponent to theless polar one was 1.107 2 0.05 for cHHAand dochrom Glucose GOD-PAP (Boehringer Mannheim) assay adaptedfor 1.509 2 0.07 for cHHB isoforms. small samples. Results from 7 animals are expressed as percent of An important indication of the position of the differences maximal hyperglycemia T standard error of the mean. RESULTS AND DISCUSSION
The existence of isoforms of the hyperglycemic hormone in different crustaceans havebeen explained by different hypotheses, such as 1)an artifactual modification during peptide purification or 2) the presence of different isoforms in different animals, related to sex or to some allelic polymorphism in the lobster population, because the peptides were purified from large pools of organs. The first hypothesis appeared unlikely, since although theprotocols used by the different investigators for extraction and purification were quite different, isoforms were observed in all cases. In order t o investigate the second hypothesis, the peptide content of single neurohemal organs from the lobster H. a m e r i c a n u s were analyzed using a micro-
between peptides was obtained from peptide mapping experiments. After hydrolysis of the four lobster hyperglycemic peptides with endoproteinase Lys-C under reducing conditions and subsequent separationof the fragmentsby HPLC, we observed (in agreement with the data in Ref. 91, that one fragment (indicated by the a r r o w in Fig. 2, left p a n e l s ) was eluted with a different retention time. This fragment wasunambiguously identified as the N-terminal octapeptide, since its molecular mass (921.0-921.6 Da), as determined by electrospray mass spectrometry (Fig. 2, right p a n e l s ) was in agreement with the theoretical average mass (921.05 Da) calculated for the protonated peptide pGlu-Val-Phe-Asp-Gln-Ala-Cys-Lys. The only remaining explanation for this change of HPLC behavior with no mass change was a modification of the chiralityof one or sev-
18297
D-Amino Acid Residue in Invertebrate Neurohormones
I
L
BB
50
.....
,.,,
10
20
40 -30 ........,. .20
30
10
20
30
10
20
30
10
7
00
RETENTIONTIME
RETENTIONTIME
(min)
40
mlz
FIG.2. Characterization of the N-terminal octapeptide of the two isoforms of cHHk Left panels,typical RP-HPLC analysisof the digestion mixture of approximately 1 nmol of the first (upper) and second (lower) isoform of cHHA by endoproteinase Lys-C under reducing conditions according toRef. 9. The arrow indicates the position of the fragment that differs between theisoforms. Chromatographic conditions were as follows. Column length was 250 mm, with a 4.6-mm internal diameter. Stationary phase wasNucleosil C-18 (5-pm particle size). Solvent A was 0.1% trifluoracetic acid in water; solvent B was acetonitrile, 0.1% trifluoracetic acid.Flow rate was 1 mumin. The elution gradient is shown by the dotted line on the upper panel. Right panels, electrospray ionization mass spectra of these fragments. The precision of the calculated mass (indicated above the maximal signal peak) is * 1 Da. The fragment eluted a t 25 min (left panels) was analyzed by electrospray ionization mass spectra and showed a (M + H)' mass of 1111.7 f 1 Da (spectra not shown), which corresponds to the mass of thefragmentcomprisingresidues 9-17 (calculatedvalue: 1111.6 Da) of the amino acid sequences in Table I.
40 (rnin)
FIG.3. Chiral amino acid analysis of hydrolysates of the Nterminal octapeptides from the first (upperpanel)and the second (lower panel)isoforms of cHHA. Chirality of the amino acids was determined by pre-column derivatization of approximately 250 pmol of acid hydrolysate with the chiral reagentFDNPA, according to Ref. 10. Chromatographic conditions were as follows. Column length was 250 mm, with a 4.6-mm internal diameter. Stationary phase was Nucleosil C-18 (5-pmparticlesize).Solvent A wastriethylamine/ phosphoric acid buffer (40 mM, pH 2.2); solvent B was acetonitrile/2propanol (4/1). Elution was realized by a convex gradient from 20% to 50%B at a flow rate of 1mumin. UV absorbance was monitoredat 340 nm (0.02 absorbance units at full scale). Identification of the derivatives was made by comparison with similar analysis of L- or D-amino acid standard solutions (not shown).
0 100 200 300 400 500 era1 amino acid residues in the cHH(1-8) fragment from one isoform as compared to the other. TIME (MIN) In order to investigate the chirality of the amino acid resiFIG.4. Differential effect of cHHA (open circles) and dues of the cHH( 1-8) peptides, a microcharacterization method [o-Phe']cHHA (filled circles)on the time course of the glycemia in the crayfish 0. limosus. Determinations of glucose level were done was used based on the derivatization of amino acids with a from 25 pl of hemolymph. Number of experimental animals was 7. chiral reagent, l-fluoro-2,4-dinitrophenyl-5-~-alanine, and sub- Results are expressed as percentof maximal hyperglycemia* standard sequent chromatographic separation (10). This procedure ap- error of the mean (vertical bars). It should be noted that the maximal plied to the cHH(1-8) peptides after acid hydrolysis showed a values of hyperglycemia obtained with both peptides were not signifiH A ,2 5.6 clear difference for the phenylalanyl residue which was found cantly different (cHHA, 30.5 * 6.7 mg/100 ml; [ D - P ~ ~ ~ I c H34.3 mg/100 ml). to be in theL-configuration in thepeptides from the more polar isoforms of cHHA and cHHB (as exemplified for cHHAin Fig. 3, Since synthetic D-amino acid-containingpeptides are curupper panel)whereas a o-configuration was present in the less rently used in pharmacology, due to their increased resistance polar isoforms (Fig. 3, lower panel). This finding was further to proteolytic enzymes, we have looked for differences in the confirmed by the chromatographic analysis of the synthetic time course of the hyperglycemic response following injection of peptides pGlu-Val-dD-Phe-Asp-Gln-Ala-Cys-Lys (chromato- the two isoforms of cHHA into crayfish. It wasobserved (Fig. 4) grams not shown). that theinjection of 40 pmol of cHHA evoked a rapid hyperglyThe same technique applied to the peptide fragment contain- cemia with a maximal value reached after 2 h, whereas the ing Phe" (see legend t o Fig. 2) showed the presence of an maximal response was attained only after 3-4 h when using L-configuration in all isoforms (chromatograms not shown). [~-phe~]cHHA. Maximal values were in the same range.MoreFrom these results, it seems that the difference between the over, the return to base-line values was slower with the latter isoforms is due toa difference in the chiralityof the phenylala- peptide. Althoughit cannot be decided from these data whether nyl residue inposition 3 of the sequence. We propose to call the these differences are connected with differences of binding conmore polar isoforms from each pair cHHA and cHHB, respec- stants to receptors and/or of half-life, it is clear that thetime tively, while the second minor ones should be named course of the effect of both isoforms is different, and the in[ ~ - p h e ~ ] c H H A a n d [ D - P ~ ~We ~ J care H Hpresently B. considering creased duration of the [ D - P ~ ~ ~ J c H effect H A fits well with availthe possibility that the same kind of structural modification able literature on synthetic D-amino acid-containing peptides. may occur in otherneurohormones, for instance in theisoforms There are only a few examples of natural peptides containing of the lobster vitellogenesis inhibiting hormone (see Fig. 1, a D-amino acid residue in animals. The first report was made in VIH),which have the same primary structure(11). the early 1980s by Montecucchi et al. (121, who described the
18298
D-Amino Residue Acid
in Invertebrate Neurohormones
presence of a D-alanine in position 2 of the opioid peptide dermorphin isolated from the skin of the frog Phyllomedusa sauvagei. The same phenomenon has been found in a number of related peptides, which constitute the dermorphinfamily (for review, see Refs. 13-15). Small neuroexcitatory peptides containing a o-amino acid residue havealso been isolated fromthe nervous system of mollusks (16). Recently, isoforms of antibacterial peptides presenting either an isoleucine or a o-alloisoleucine residue have been isolated from the skin of Bombina variegata (17).A common feature of the o-amino acid residue containing peptides characterized so far is thepresence of the D-residue in position 2 of the sequence. In this respect, lobster cHHs differ, since the D-residue occurs in position 3, but it is still very close t o the N-terminal extremity. a matter of debate. The origin of D-aminoacid residues is still However, the hypothesis of a post-translational modification of an L-residue into its enantiomer appears the most plausible: in the dermorphin precursor, the classical GCG alanine codon is found in the position where D-alanine is present in the final product (18).At the present timeit is not known whether both isoforms of lobster cHH have the same precursor or if they are synthesized by the same or different neurosecretory cells; t o answer these important questions will require the design of molecular and immunological tools specific for each isoform. The presence of isoforms sharing different biological effects suggests that the conformational change of an amino acid residue, which may escape classical investigation methods, could represent an authentic (and perhaps widespread) post-transla-
tional mechanism thatgenerates single amino acid sequences.
hormonaldiversity
from
Acknowledgments-We thankProf. R. KellerandDr. D. Bocking (Bonn, Germany) for the kind provisionof crayfish used for bioassays.
REFERENCES 1. Keller, R. (1992) Experientia (Basel) 4 8 , 4 3 9 4 3 2. Newcomb, R. W.(1983) J. Comp. Physiol. 153, 207-221 3. Keller, R., and Kegel, G. (1984) in Biosynthesis, Metabolism andMode ofAction of Invertebrate Hormones (Hoffman,J., and Porchet, M., eds) pp 145-154, Springer-Verlag, Heidelberg 4. Huberman, A., and Aguilar, M. B. (1986) Comp. Biochern. Physiol. 8SB, 197203 5. Soyez, D., Van Deijnen, J. E., and Martin, M. (1987)J. Exp. Zool. 244,479-484 6. Tensen, C . P., Janssen, K. P. C., and Van Herp, F.(1989) Invert. Reprod. Dev. 16, 155-164 7. Tensen, C. P., De Kleijn, D. P. V., and Van Herp,F.(1991)Eur:J. Biochem. 200, 103-106 8. Soyez, D., Noel, P. Y., Van Deijnen, J. E., Martin, M., Morel, A., and Payen, G. G. (1990) Gen. Cornp. Endocrinol. 79, 261-274 9. Tensen, C . P., Janssen, K. P. C., Soyez, D., and Van Herp, F.(1991)Peptides 12, 241-249 10. Scaloni, A., Simmaco, M., and Bossa, F.(1991) Anal. Biochem. 197, 305310 11. Soyez, D., Lecaer, J. P., Noel, P. Y., and Rossier, J. (1991) Neuropeptides 20, 2532 12. Montecucchi, P.C., De Castiglione, R., Piani, S., Gozzini, L., and Erspamer,V. (1981) Int. J. Peptide Protein Res. 17, 275-283 13. Erspamer, V. (1992) Int. J . Deu. Neurosci. 10, 3-30 14. Mor, A., Amiche M., and Nicolas, P. (1992) 'Dends Biochern. Sci. 17,481-485 15. Lazarus, L. H., and Attila M. (1993) Prog. Neurobiol. 41, 473-507 16. Muneoka, Y., and Kobayashi, M. (1992) Experientia (Basel)48, 448-456 17. Mignogna, G., Simmaco, M., Kreil, G., and Barra, D. (1993) EMEO J . 12, 4829-4832 18. Richter, K,, Egger, R., and Kreil, G. (1987) Science 238,200-202
