GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
81,410-418
(1991)
Immunological Relationships between Neuropeptides from the Sinus Gland of the Lobster Homarus americanus, with Special References to the Vitellogenesis Inhibiting Hormone and Crustacean Hyperglycemic Hormone JEAN-JACQUES
MEUSY
AND DANIEL
SOYEZ’
Laboratoire de Physiologie de la Reproduction, Equipe de Neuroendocrinologie des CrustacCs, Universitk Pierre et Marie Curie et CNRS UA 040555, B&iment A, 4 place Jussieu, F75252 Paris Cedex 05, France Accepted April 6, 1990 Antisera raised in guinea pigs against four major neuropeptides purified from sinus glands of the lobster, Homarus americanus, were used to study the immunological relationships between several sinus gland peptides. On the basis of their behavior in ELISA and in absorption procedures, three groups of peptides are defined. Two groups may be related to the crustacean hyperglycemic hormone (CHH groups); the third one is composed of three immunologically identical peptides and, since one of these peptides was characterized in previous studies as a vitellogenesis inhibitor, is referred to as VIH group. This closely meets our present knowledge about the physiological effects and biochemical characteristics of these neuropeptides and gives immunological insights on the question of molecular polymorphism of lobster neurohormones. 8 1991Academic press, IIIC.
In the eyestalks of Malacostracan Crustacea, a cluster of neurosecretory cells (Xorgan) elaborates a variety of neuropeptides which accumulate in a neurohaemal organ (the sinus gland, SG) before their release within the hemolymph. These neurohormones control physiological processes such as color adaptation (chromatotropic hormones), glycemia (crustacean hyperglycemic hormone, CHH), molt (molt-inhibiting hormone, MIH), reproduction (vitellogenesis inhibiting hormone, VIH; also called gonad inhibiting hormone, GIH) (reviews in Kleinholz and Keller, 1979; Meusy and Payen, 1988). Fractionation of lobster sinus gland extract by RP-HPLC showed that the major neuropeptides (numbered 1 to 6) are eluted in three sets of two peptides of close hydro-
phobicity (Soyez et al., 1987). Among them, peptide 3 was characterized as the vitellogenesis inhibitor, using a heterologous bioassay developed in eyestalkless shrimps Palaemonetes varians. This peptide, assimilated to the lobster VIH, has an apparent molecular weight close to 7.5 kDa, as estimated by SDS-PAGE. Preliminary biochemical analysis (Soyez et al., 1988) indicated strong similarities in amino acid composition and pHi of the paired peptides (1 and 2, 3 and 4, and 5 and 6). In a previous study, a mouse antiserum was raised against peptide 3 (VIH) and its specificity was studied by immunochemistry and immunocytochemistry (Meusy et al., 1987). VIH was selectively recognized among the major lobster SG peptides in enzyme linked immunosorbent assays (ELISA) and one neurosecretory granule type was labeled by this antiserum on SG sections. Using the anti-VIH serum together with an antiserum raised against the hyperglycemic hormone of the crayfish As-
’ To whom correspondence should be addressed at present address: Laboratoire de Biochimie CNRS UA 686, Ecole Normale Superieure 46 rue d’Ulm, 75230 Paris cedex 05, France. 410 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All tights of reproduction in any form reserved.
CRUSTACEAN
NEUROHORMONES
tacus leptodactylus, Kallen and Meusy (1989) described the respective location of VIH and CHH in Homarus neurosecretory structures. In the SG, both neuropeptides are mostly located in different neuronal endings containing distinct granule types. In the X-organ, some perikarya are labeled by both antisera, while some others display a positive immunoreaction with only one antiserum (anti-CHH or anti-VIH). These results could suggest the existence of a common precursor for both hormones. In order to elucidate relationships between the different Homarus neuropeptides, and to develop specific tools for further studies, we prepared antisera against purified peptides 1,3,4 (biochemically similar to 3), and 5. These antisera were raised in guinea pigs. Their specificity was determined. Immunological relationships between the different SG peptides were further investigated by selective absorption experiments of anti-3 and anti-4 antibodies. MATERIAL AND METHODS Eyestalks from adult Homarus were obtained from the coast of New Brunswick (Canada). They were stored freeze-dried at -80”. Approximately 4,600 eyestalks were used for peptide preparation. Before dissection, eyestalks were rehydrated in ice cold water. SG were ground in 10% acetic acid (1 ml/80 SG) and the homogenate was maintained in a water bath at 85” during 5 min. After centrifugation (15,OOOg, 20 min), the pellet was reextracted with 1 ml acetic acid and centrifuged. The pooled supematants were ultracentrifuged (165,OOOg, 15 min at 4”). The supematant was used immediately for HPLC or stored frozen until use. Occasionally, SG peptides were extracted with acetic acid at room temperature or at 4”. The chromatographic equipment was a LKB low pressure gradient system, the eluants being degassed by a continuous flow of helium; uv detection was realized at 280 nm with a Pharmacia UV 2 detector. Analytical chromatographies were performed on a Nucleosil C-18 column (5 urn particle size, 25 cm length X 0.46 cm internal diameter), with a discontinuous linear gradient of n-propanol/O. 1% trifluoracetic acid (TFA) in water/O. 1% TFA (pH 2.2). at a flow rate of 0.75 ml/min. Large-scale preparation of peptides involved two steps of HPLC in different eluant systems (pH 6.2 then pH 2.2): in a first step of purification, SG extract (80Peptide americanus
purification.
411
100 SG equivalents) was fractionated on a Nucleosil C-18, 5-urn particle-size column (25 cm length x 0.46 cm i.d.), with a linear gradient (10 to 50%) of acetonitrile (l%/min) in phosphate buffer 25 n-&f, pH 6.2. Flow rate was 1 mumin. Fractions were collected for 30 set in Nunc Minisorp polyethylene tubes and then dried under vacuum with a Speed-Vat centrifuge concentrator (Savant). In a second step, homologous fractions from different preparations were pooled and rechromatographed in the n-propanol/O. 1% TFA/water solvent system (pH 2.2) on a Vydac C4 wide pore column (15 cm x 0.46 cm i.d.) or a Nucleosil C-18, 5-urn particle-size cartridge (12.5 cm x 0.46 cm). In order to avoid cross contaminations, a Nucleosil cartridge was used for the purification of one group of peptides only. After vacuum drying of pooled fractions, the peptides were stored under nitrogen at - 20”. Peptide content of selected fractions was quantified by the method of Bradford (1976) and related to peak area on the chromatogram. Subsequently, peptide amount was deduced from the corresponding peak area. Immunization procedure. The immunizations were carried out on Dunkin Hartley guinea pigs, at CNRZINRA Station (Jouy-en-Josas, France). Before any injection, the guinea pig sera were controlled for their absence of reactivity against sinus gland extract. The animals were numbered by an ear clip. Four purified neuropeptides (1, 3, 4, and 5) were injected in two animals each. The pairs of animals were kept in separate cages. The same peptide preparation was used for all the immunization process. For the first injection, 20 ug of peptide was dissolved in sterile physiological saline and emulsified with complete Freund’s adjuvant. The subsequent injections (10 ug of peptide in incomplete Freund’s adjuvant) were made one every 2 weeks for 6 weeks. The first three injections were made subcutaneously at multiple sites along the back and the fourth one was made intraperitoneally. Eight days after the last injection, the blood was collected and the sera were tested. Five weeks later, a booster of 5 ug of peptide was given subcutaneously. Twelve days later, all animals were bled and the homologous sera were pooled. The crude y-globulins were precipitated by ammonium sulfate (33%) redissolved in 50 mM phosphate buffer, pH 7.2, and dialyzed against the same buffer. The volume of the Ig fraction was adjusted with phosphate buffer to the initial volume of the serum. ELISA. Direct ELISA was performed on microtiter plates (Nunc) coated with purified peptides (40 ng of antigen per well in 0.1 ml sodium carbonate buffer, 0.1 M, pH 9.6) for 2 hrs at 37” or one night at 5”. After three washings in 0.02 M PBS, pH 7.4 (containing 0.1% Tween 20 and 0.02% sodium azide), the plates were incubated for 1.5 hr at 37” with guinea pig antibodies diluted 1:500 or I:300 in PBS buffer containing 1% normal goat serum. After three washings, a new incubation performed (1.5 hr at 37”) with an anti-
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guinea pig IgG alkaline phosphatase conjugate developed in goat (Sigma A-7664), diluted 1:lOOO. Finally, the enzymatic activity was assessed by the addition of the substrate (1 mg/ml p-nitrophenyl phosphate disodium in 0.05 M carbonate buffer, pH 9.8). The reaction was not stopped and the optical density was determined every 10 min at 410 nm using a Dynatech ELISA reader. Absorption of antibodies. Increasing amounts of peptides, from 0 to 25 CLp,were each redissolved in 15 ul of antibodies diluted with 45 u1 of PBS (50 mM, pH 7.2). The tubes were incubated 1 hr at 37” and one night at 5”, and centrifuged at 20,OOOgfor 20 min. The supematants were diluted to obtain a final dilution of l/300 or l/500 for all samples. The ELISA was carried out as before.
RESULTS Immunoreactivity of the Antibodies against the Major Lobster Sinus Gland Peptides Figure 1 shows a typical elution pattern resulting from the fractionation of hot acetic acid sinus gland extract in the analytical propanol/water/TFA HPLC system. The major peptides, eluted between 24 and 26% of n-propanol, are numbered 1 to 6 according to their elution positions. The shoulder between 3 and 4 (numbered 3’) appears consistently. The amount extractable from one SG was estimated as 250 ng for peptide I,80 ng for peptide 2,55 ng for peptide 3,95 ng for peptide 4, 260 ng for peptide 5, and 210 ng for peptide 6.
SOYEZ
After extraction in cold acetic acid, two extra peaks (numbered 7 and 8) with almost the same retention times as peaks 1 and 2 were present, in addition to more polar peptides eluted in earlier zone of the gradient (Fig. 2). A lengthy extraction (1.5 to 2 hr) of SG homogenate in cold acid often results in the disappearance of peaks 1, 2, 5, and 6. Peaks 3, 3’, and 4 are not modified (chromatogram not shown). Peaks 7, 8, and 3’, as well as the major peptides 1 to 6, have been included in the immunological studies reported below. Determination of the immunoreactivity of anti-3 and anti-4 antibodies by ELBA shows a positive reaction with all peptides tested (Table 1). Maximal reactions are obtained with peptides 3’ and 4, then with 3. Anti-l and anti-5 antibodies do not recognize peptides 3, 3’, and 4 and display the strongest responses with peptides 1 and 2 (Table 1). As mentioned on Table 1, some variations of the reactivity are observed from one experiment to another-and from one peptide preparation to anotherwithout modifications of the general features of the responses. The results described above indicate that anti-3 and anti-4 antibodies display a poor specificity toward the different SG peptides. In order to investigate whether this observation resulted from the presence of
I..% propanol
115
t (min) FIG. 1. RP-HPLC elution profile of lobster sinus glands extracted by hot acetic acid. Thirty sinus glands were extracted during 5 min in 10% acetic acid at 85”. Column, Nucleosil C18, 5 urn (25 cm x 0.46 cm i.d.); solvents, 0.1% TFA in water and 0.1% TFA in n-propanol. Flow rate, 0.75 ml/min. The gradient of n-propanol is indicated by the dotted line. AUFS, absorbance unit full scale; t, time of elution in minutes. The major peaks are numbered I to 6. Peak 3 was characterized as vitellogenesis inhibitor (VIH) in previous studies (see the introduction). 0
10
20
30
40
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.....% propanol 40 6
AUFS 0,002 I .-’
,_._._._._._.__... .............. .:’ ,.I’ ._.’ ,..’ 1” ..’ _____._. II:.’...’
J
20
10
0
30
40
I’
30
:
20
t (mn)
FIG. 2. RP-HPLC profile of lobster sinus glands extracted by cold acetic acid. Forty sinus glands were extracted in 10% acetic acid at room temperature during 30 min. Column, Nucleosil C18, 5 pm (25 cm x 0.46 cm i.d.); solvents, 0.1% TFA in water and 0.1% TFA in n-propanol. Flow rate, 0.75 mk’min. The gradient of n-propanol is indicated by the dotted line. AUF& absorbance unit full scale; t, time of elution in minutes. The main difference with the chromatogram presented in Fig. 1 is the presence of peaks 7 and 8, eluted around 30 min.
several types of IgG in the antibodies preparations or from the existence of common epitopes on the different peptides, absorptions of these antibodies by the reacting peptides were realized, followed by ELISA determination of the remaining immunoreactivity. Absorption of Anti-3 Antibodies Peptide 5 and Peptide 7
by
When 15 J.L~of anti-3 antibodies
are abTABLE
IMMUNOREACTIVITY
sorbed by increasing amounts of peptide 5, the reactivity of the remaining immunoglobulins with peptides 3, 3’, and 4 is not affected (Fig. 3). In contrast, absorption with 250 ng of peptide 5 is sufficient to decrease at least fivefold the reaction with peptides 1, 2, 5, 6, 7, and 8. With peptides 1 and 2 only, a reaction is still present after absorption with 5 kg of peptide 5. Similar results are obtained when anti-3 antibodies are absorbed by peptide 7 (Fig. 4). Absorptions of anti-3 antibodies by 1
OF ANTIBODIES RAISED AGAINST FOUR PEPTIDES FROM Homarus americanus SINUS GLANDS
PURIFIED
BY
HPLC
Antibody Peptide
Anti-l
Anti-3
Anti-4
Anti-5
1
+ +I+ + +
2 3 3' 4 5 6 7 8
+++ 0 0 0 + + +I-?- -t -k-k
++
++
++ + +I+ + + -I- +I-!- + + + + +I-+ + + + +I+ + + ++ ++
++ + -k/i- + + + +I+ + + + + +I+ + + + + + + +
++++ ++++ 0 0 0 + +I+ + + + ++I+++ +++ +++
Note. The antibodies were tested in ELISA against the homologous antigens (bold figures) and the other major sinus gland neuropeptides. The relative immunoreactivity is expressed from 0 to + + + + .
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MEUSY
AND
SOYEZ 1.5 -
E
1.0.
z 2
0.5
01
2
3
3’ 4 PEPTIDE
5
6
7
1
8
FIG. 3. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 5, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 t.~l of undiluted antibodies. Optical density values correspond to 30-min readings.
higher amounts of peptide 5 were carried out in order to determine if the immunoreactivity with peptide 1 (or 2) could be completely suppressed. As shown in Fig. 5, this reactivity remains even when 25 pg of peptide are used for absorbing 15 p,l of antibodies solution. In these conditions, the reactivity with peptide 3 is not significantly reduced . Absorption of Anti-3 and Anti-4 Antibodies by Peptide 2 Figure 6 shows that a small amount of peptide 2 (250 ng/lS p,l antibodies solution)
3
5
7
PEPTIDE
FIG. 5. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with large quantities of peptide 5, as revealed by ELISA against peptides 1,3,5, and 7. In the insert: amount of peptide added to 15 l.d of undiluted antibodies. Optical density values correspond to 20-min readings.
suppresses the reaction of anti-3 antibodies with peptides 1 and 2, while the reactivity for peptides 5,6, 7, and 8 is decreased by a twofold factor. Increased amounts of antigen do not significantly modify the response. No significant modification of the response with peptides 3, 3’, and 4 is observed in this experiment. Similar features are observed when the ELISA is realized with anti-4 antibodies (Fig. 7), though the reactivity of these antibodies with 1 and 2 peptides cannot be totally suppressed. Absorption of Anti-3 Antibodies Peptides 2 + 5 When the anti-3 antibodies
by are simulta-
Is-
E E
1.0-
z 2
0.5.
3
3’
4 PEPTI
5
6
7
8
DE
FIG. 4. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 7, as revealed by ELBA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 pl of undiluted antibodies. Optical density values correspond to 30-min readings.
1
2
3
3’
4
5
6
7
8
PEPTIDE
FIG. 6. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 2, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ~1 of undiluted antibodies. Optical density values correspond to 20-min readings.
CRUSTACEAN
415
NEUROHORMONES I.00
0.80
.
E c
Peptlde
Peptlde 3
I
0
Pqmde
5
.
Peptide
1
0.60
s
;
I
=
l-----l 0.40
.
.
, 0
.
.
l -2
0.20
2
3
3’
4
s
6
8
7
PEPTIDE
7. Immunoreactivity of anti-4 antibodies witbout absorption and after absorption with increasing quantities of peptide 2, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ul of undiluted antibodies. Optical density values correspond to 20min readings. FIG.
neously absorbed by equal amounts of peptides 2 and 5, the response with peptides 1, 2, 5, 6, 7, and 8 is completely abolished (Fig. 8). The reaction with 3, 3’, and 4 is unaffected, whatever be the quantity of 2 + 5 peptides added. Absorption of Anti-3 Antibodies Peptide 4
by
Immunoreactivity of anti-3 antibodies has been investigated after absorption with increasing amounts of peptide 4 (Fig. 9). A
0 1
2
3
3’
4
5
6
7
8
PEPTIDE
FIG. 8. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 2 + peptide 5, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ul of undiluted antibodies. Optical density values correspond to 25min readings.
0
10’ 10’ 10’ ABSORPTION (ng of peptide)
10’
FIG. 9. ELBA absorption profiles of anti-3 (antiVIH) antibodies absorbed by increasing amounts of peptide 4 and tested against peptides 1, 3, 5, and 7. Abscissa represents the amount of peptide 4 used for absorption on a logarithmic scale. Optical density values correspond to 20-min readings.
sigmoidal absorption curve is obtained when the antibodies are tested against peptide 3. The reaction is completely abolished when 15 l.rl of anti-3 antibodies are absorbed with 5 and 10 p.g of peptide 4. In contrast, the reactivity against peptides 5 and 7 is not modified whereas a slight reduction of the response is observed with peptide 1 when 5 or 10 p,g of antigen 4 is added to the antibodies. DISCUSSION
The present investigation on the specificity of guinea pig antisera raised against purified lobster neuropeptides indicates the existence, in these molecules, of some common immunological characteristics. ELISA experiments realized with antibodies raised against peptide 1 and against peptide 5 indicates that peptides 1, 2, 5, 6, 7, and 8 are recognized by both antibodies and therefore do possess common antigenic determinants. On the other hand, peptides 3 and 4 (as well as 3’) do not react with the anti-l and anti-5 antibodies and are highly recognized by anti-3 and anti-4 antibodies (peptide 4 being generally more reactive). As a matter of fact, all the nine peptides tested (i.e., 1, 2, 3, 3’, 4, 5, 6, 7, and 8) are recognized by anti-3 (or anti-4) antibodies.
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After selective absorptions, however, the anti-3 antibodies become strictly specific for peptides 3, 3’, and 4. Therefore, these peptides seem closely related. This result reinforces and completes previous work which indicates that peptide 3, biologically characterized as VIH (Soyez et al., 1987), and peptide 4 may be structurally related since they display analogous amino acid composition and pHi. (Soyez et al., 1988). In a previous study, antisera were raised in Biozzi mice against peak 3. Two out of three mouse antisera reacted slightly with peak 1 peptide. After absorption with this peptide the antisera recognized only peak 3. In no case did the mouse antisera crossreact with peptide 4 (Meusy ef al., 1987). The difference with the results exposed in the present report could be ascribed to the immunization itself which was very light in the experiment with mice: 7.7 kg of peptide per mouse in three injections as compared to 55 pg per guinea pig in five injections for the present study. The absorption of anti-3 antibodies by peptide 5 (or 7) completely abolished the reaction with peptides 5, 6, 7, and 8. Reactivity with peptides 1 and 2 strongly decreased when increasing amounts of peptide 5 were used, but could not be completely suppressed, while reaction with peptides 3, 3’) and 4 was not affected. This result is very surprising since the antibodies were not supposed to be raised against any other epitopes than those of the injected peptide 3. A likely hypothesis could be that the epitopes involved in the absorptions are not present-or not reactant-in those three last peptides. A trivial error during preparation or injections is highly improbable since strict precautions have been taken; furthermore, it must be recalled that antibodies against peptides 1 and 5 did not react with peptides 3, 3’ and 4 (it would not be the case if some animals had been mixed). It is likely that an epitope of peptide 3, also present in peptides 5 and 7, was immunogenic upon injections in guinea pigs
SOYEZ
but was not antigenic in ELISA determinations or during absorption processes. This epitope could be masked in native peptide 3 (or 4) and would be accessible during immunization. Such a hypothesis agrees with recent immunological theories where the cleavage of antigens within endosomes of B-lymphocytes is described as an early step of immunization process, followed by the raising of antibodies against some peptidic fragments (review in Long, 1989). The existence of a common epitope present in the major sinus gland peptides and masked in native peptides 3 or 4 should be proved by demonstrating the existence of structural homologies between all these peptides. The absorption of anti-3 and anti-4 peptides by peptide 2 gave results comparable to those obtained after absorptions with peptides 5 or 7: the reactivity with peptides 1 and 2 was suppressed, the response with peptides 5, 6, 7, and 8 was only decreased down to some steady level, while reactivity with peptides 3, 3’, and 4 was not changed. So, we can postulate that the epitopes of peptides 1 and 2 are completely different from the reactant epitopes of peptides 3,3’, and 4 and are partially common with those of peptides 5, 6, 7, and 8. This agrees with recent data which indicates that the four peptides 1, 2, 5, and 6 display a hyperglycemic activity though the first two peptides present a different isoelectric point, molecular mass, and amino acid composition from the last two (Soyez et al., 1990). As expected, after absorptions of anti-3 antibodies by 2 + 5 peptides, only peptides 3, 3’, and 4 are recognized. After absorption of anti-3 antibodies by peptide 4, their reaction with peptide 3 falls down to zero, following a standard sigmoidal curve. This result confirms that the reacting epitopes of peptides 3 and 4 are identical, while those of peptides 1, 5, and 7 seem different from them. When repeating ELISA experiments, quantitative variations of reactivity were often observed. Since aliquots of the same
CRUSTACEAN
417
NEUROHORMONES
antibody preparations were used each time, this feature may be attributed to the antigens themselves. Small inaccuracies in quantification and solubilization can contribute to these variations by physicochemical modification(s) during storage, such as oxidation of methionine residues for example, can also be involved. We frequently observed that frozen and thawed antigens are more reactive than those freshly prepared. This could be related to the existence of some masked epitope in native peptides, as discussed above. Peptides 7 and 8 were observed only after extraction of SG in acetic acid in cold or at room temperature, and their presence is correlated with a reduction of the amount of peptides 5 and 6. This observation agrees with the results of Chang et al. (1987): these authors characterized a doublet peak with molt-inhibiting activity (peak 8) from the SG of the lobster H. americanus. These peptides slowly degrade in cold acetic acid, generating shorter peptides (doublet peak 5) which lack bioactivity. On the basis of chromatographic behaviour and amino acid composition analyses, Soyez et al. (1990) suggest that hyperglycemic peptides 5 + 6 are homologous to doublet peak 8 from Chang et al. (1987). Therefore doublet peak 5 described by these last authors could correspond to peptides 7 + 8 studied in the present report. Close structural relationships between peptides 5 + 6 and 7 + 8 are further confirmed by the identical immunological behaviour of all these peptides in the absorption experiments of anti-3 antibodies. As mentioned above, our experiments indicate that peptides 1, 2, 5, 6, 7, and 8 display at least one identical epitope. Furthermore, CHH peptides (as well as VIH) (Meusy et al., 1987) are not strictly species specific. Previous results obtained by dot immunobinding assay (DIA) or ELISA suggest that hyperglycemic peptides from other crustacean species may display the same epitope: an antisera raised against
CHH from the crayfish A. leptodactylus’ recognized the lobster CHH related peptides (Van Deijnen, 1986; Tensen et al., 1989 and Meusy, unpublished results). The same observation was made with an antisera raised against CHH from Orconectes limosus* (Meusy, unpublished results). Therefore structural analogies may exist between hyperglycemia inducing peptides within, at least, the astacidea. To conclude, among the HPLC-purified peptides from the lobster sinus gland, three immunological families can be distinguished: the peptides 1, 2 group (CHH group number 1); the peptides 5, 6, 7, 8 group (CHH group number 2); and the peptides 3, 3’, 4 group (VIH group). These groups do present some identical antigenic determinant(s) and it will now be interesting to elucidate the structural relationships between these peptides in relation to their synthesis pathway and physiological significance. ACKNOWLEDGMENTS The authors thank Dr. Gerard Conan and Ms. Claire Doucette for having supervised the supply of lobster eyestalks and Mr. Moret and the staff of CNRZ-INRA for their assistance during the immunization procedures. We are grateful to Ms. Madeleine Martin for her technical collaboration and to our colleagues for their helpful participation in the preparation of the sinus glands. We thank Professor J. Charlemagne for his critical reading of our manuscript.
REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Chang, E. S., Bruce, M. J., and Newcomb, R. W. (1987). Purification and amino acid composition of a peptide with molt-inhibiting activity from the lobster, Homarus americanus. Gen. Comp. Endocrinol. 65, 56-64.
’ From Dr. F. Van Herp. ’ From Professor R. Keller.
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Kallen, J., and Meusy, J.-J. (1989). Do the neurohormanes VIH (Vitellogenesis Inhibiting Hormone) and CHH (Crustacean Hyperglycemic Hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster, Homarus americanus. Invertebr. Reprod. Dev. 16, 43-52. Keller, R. (1969). Untersuchungen zur artspezititlt eines crustaceenhormons. 2. Vgl. Physiol. 63, 137-14s. Keller, R. (1977). Comparative electrophoretic studies of crustacean neurosecretory hyperglycemic and melanophore-stimulating hormones from isolated sinus glands. J. Comp. Physiol. 122, 359-373. Kleinholz, L. H., and Keller, R. (1979). Endocrine regulation in Crustacea. In “Hormones and Evolution” (E. J. W. Barrington, Ed.), Vol. 1, pp. 159-213. Academic Press, New York. Leuven R. S. E. W., Jaros, P. P., Van Herp, F., and Keller, R. (1982). Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. Gen. Camp. Endocrinol. 46, 288-296. Long, E. 0. (1989). Intracellular traftic and antigen processing. Immunol. Today 10, 232-234. Meusy, J.-J., Martin, G., Soyez, D., Van Deijnen, J. E., and Gallo, J.-M. (1987). Immunochemical and immunocytochemical studies of the crustacean vitellogenesis-inhibiting hormone. Gen. Comp. Endocrinol. 55, 208-216.
Meusy, J.-J., and Payen, G. G. (1988). Female reproduction in Malacostracan crustacea. Zool. Sci. 5, 217-265. Soyez, D., Noel, P. Y., Van Deijnen, J. E., Martin, M., Morel, A., and Payen, G. G. (1990). Neuropeptides from the sinus gland of the lobster Homarus americanus. Characterization of hyperglycemic peptides. Gen. Comp. Endocrinol. 79, 261-274. Soyez, D., Van De&ten, J. E., Laverdure, A. M., Martin, M., Meusy, J.-J., Noel, P. Y., Payen, G. G., Tensen, C. P., and Van Herp, F. (1988). Neuropeptides from the sinus gland of the lobster Homarus americanus. Gen. Comp. Endocrinol. 74, 319 [Abstract]. Soyez, D., Van Deijnen, J. E., and Martin, M. (1987). Isolation and characterization of a vitellogenesisinhibiting factor from sinus glands of the lobster, Homarus americanus. J. Exp. Zoo/. 244,479-484. Tensen, C. P., Janssen, K. P. C., and Van Herp, F. (1989). Isolation, characterization and physiological specificity of the crustacean hyperglycemic factors from the sinus glands of the lobster, Homarus americanus (Milne-Edwards). Invertebr. Reprod. Dev. 16, 155-164. Van Deijnen, J. E. (1986). ‘Structural and Biochemical Investigations into the Neuroendocrine System of the Optic Ganglia of Decapod Crustacea.” Ph.D. Thesis, Catholic University, Nijmegen, The Netherlands.
AND
COMPARATIVE
ENDOCRINOLOGY
81,410-418
(1991)
Immunological Relationships between Neuropeptides from the Sinus Gland of the Lobster Homarus americanus, with Special References to the Vitellogenesis Inhibiting Hormone and Crustacean Hyperglycemic Hormone JEAN-JACQUES
MEUSY
AND DANIEL
SOYEZ’
Laboratoire de Physiologie de la Reproduction, Equipe de Neuroendocrinologie des CrustacCs, Universitk Pierre et Marie Curie et CNRS UA 040555, B&iment A, 4 place Jussieu, F75252 Paris Cedex 05, France Accepted April 6, 1990 Antisera raised in guinea pigs against four major neuropeptides purified from sinus glands of the lobster, Homarus americanus, were used to study the immunological relationships between several sinus gland peptides. On the basis of their behavior in ELISA and in absorption procedures, three groups of peptides are defined. Two groups may be related to the crustacean hyperglycemic hormone (CHH groups); the third one is composed of three immunologically identical peptides and, since one of these peptides was characterized in previous studies as a vitellogenesis inhibitor, is referred to as VIH group. This closely meets our present knowledge about the physiological effects and biochemical characteristics of these neuropeptides and gives immunological insights on the question of molecular polymorphism of lobster neurohormones. 8 1991Academic press, IIIC.
In the eyestalks of Malacostracan Crustacea, a cluster of neurosecretory cells (Xorgan) elaborates a variety of neuropeptides which accumulate in a neurohaemal organ (the sinus gland, SG) before their release within the hemolymph. These neurohormones control physiological processes such as color adaptation (chromatotropic hormones), glycemia (crustacean hyperglycemic hormone, CHH), molt (molt-inhibiting hormone, MIH), reproduction (vitellogenesis inhibiting hormone, VIH; also called gonad inhibiting hormone, GIH) (reviews in Kleinholz and Keller, 1979; Meusy and Payen, 1988). Fractionation of lobster sinus gland extract by RP-HPLC showed that the major neuropeptides (numbered 1 to 6) are eluted in three sets of two peptides of close hydro-
phobicity (Soyez et al., 1987). Among them, peptide 3 was characterized as the vitellogenesis inhibitor, using a heterologous bioassay developed in eyestalkless shrimps Palaemonetes varians. This peptide, assimilated to the lobster VIH, has an apparent molecular weight close to 7.5 kDa, as estimated by SDS-PAGE. Preliminary biochemical analysis (Soyez et al., 1988) indicated strong similarities in amino acid composition and pHi of the paired peptides (1 and 2, 3 and 4, and 5 and 6). In a previous study, a mouse antiserum was raised against peptide 3 (VIH) and its specificity was studied by immunochemistry and immunocytochemistry (Meusy et al., 1987). VIH was selectively recognized among the major lobster SG peptides in enzyme linked immunosorbent assays (ELISA) and one neurosecretory granule type was labeled by this antiserum on SG sections. Using the anti-VIH serum together with an antiserum raised against the hyperglycemic hormone of the crayfish As-
’ To whom correspondence should be addressed at present address: Laboratoire de Biochimie CNRS UA 686, Ecole Normale Superieure 46 rue d’Ulm, 75230 Paris cedex 05, France. 410 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All tights of reproduction in any form reserved.
CRUSTACEAN
NEUROHORMONES
tacus leptodactylus, Kallen and Meusy (1989) described the respective location of VIH and CHH in Homarus neurosecretory structures. In the SG, both neuropeptides are mostly located in different neuronal endings containing distinct granule types. In the X-organ, some perikarya are labeled by both antisera, while some others display a positive immunoreaction with only one antiserum (anti-CHH or anti-VIH). These results could suggest the existence of a common precursor for both hormones. In order to elucidate relationships between the different Homarus neuropeptides, and to develop specific tools for further studies, we prepared antisera against purified peptides 1,3,4 (biochemically similar to 3), and 5. These antisera were raised in guinea pigs. Their specificity was determined. Immunological relationships between the different SG peptides were further investigated by selective absorption experiments of anti-3 and anti-4 antibodies. MATERIAL AND METHODS Eyestalks from adult Homarus were obtained from the coast of New Brunswick (Canada). They were stored freeze-dried at -80”. Approximately 4,600 eyestalks were used for peptide preparation. Before dissection, eyestalks were rehydrated in ice cold water. SG were ground in 10% acetic acid (1 ml/80 SG) and the homogenate was maintained in a water bath at 85” during 5 min. After centrifugation (15,OOOg, 20 min), the pellet was reextracted with 1 ml acetic acid and centrifuged. The pooled supematants were ultracentrifuged (165,OOOg, 15 min at 4”). The supematant was used immediately for HPLC or stored frozen until use. Occasionally, SG peptides were extracted with acetic acid at room temperature or at 4”. The chromatographic equipment was a LKB low pressure gradient system, the eluants being degassed by a continuous flow of helium; uv detection was realized at 280 nm with a Pharmacia UV 2 detector. Analytical chromatographies were performed on a Nucleosil C-18 column (5 urn particle size, 25 cm length X 0.46 cm internal diameter), with a discontinuous linear gradient of n-propanol/O. 1% trifluoracetic acid (TFA) in water/O. 1% TFA (pH 2.2). at a flow rate of 0.75 ml/min. Large-scale preparation of peptides involved two steps of HPLC in different eluant systems (pH 6.2 then pH 2.2): in a first step of purification, SG extract (80Peptide americanus
purification.
411
100 SG equivalents) was fractionated on a Nucleosil C-18, 5-urn particle-size column (25 cm length x 0.46 cm i.d.), with a linear gradient (10 to 50%) of acetonitrile (l%/min) in phosphate buffer 25 n-&f, pH 6.2. Flow rate was 1 mumin. Fractions were collected for 30 set in Nunc Minisorp polyethylene tubes and then dried under vacuum with a Speed-Vat centrifuge concentrator (Savant). In a second step, homologous fractions from different preparations were pooled and rechromatographed in the n-propanol/O. 1% TFA/water solvent system (pH 2.2) on a Vydac C4 wide pore column (15 cm x 0.46 cm i.d.) or a Nucleosil C-18, 5-urn particle-size cartridge (12.5 cm x 0.46 cm). In order to avoid cross contaminations, a Nucleosil cartridge was used for the purification of one group of peptides only. After vacuum drying of pooled fractions, the peptides were stored under nitrogen at - 20”. Peptide content of selected fractions was quantified by the method of Bradford (1976) and related to peak area on the chromatogram. Subsequently, peptide amount was deduced from the corresponding peak area. Immunization procedure. The immunizations were carried out on Dunkin Hartley guinea pigs, at CNRZINRA Station (Jouy-en-Josas, France). Before any injection, the guinea pig sera were controlled for their absence of reactivity against sinus gland extract. The animals were numbered by an ear clip. Four purified neuropeptides (1, 3, 4, and 5) were injected in two animals each. The pairs of animals were kept in separate cages. The same peptide preparation was used for all the immunization process. For the first injection, 20 ug of peptide was dissolved in sterile physiological saline and emulsified with complete Freund’s adjuvant. The subsequent injections (10 ug of peptide in incomplete Freund’s adjuvant) were made one every 2 weeks for 6 weeks. The first three injections were made subcutaneously at multiple sites along the back and the fourth one was made intraperitoneally. Eight days after the last injection, the blood was collected and the sera were tested. Five weeks later, a booster of 5 ug of peptide was given subcutaneously. Twelve days later, all animals were bled and the homologous sera were pooled. The crude y-globulins were precipitated by ammonium sulfate (33%) redissolved in 50 mM phosphate buffer, pH 7.2, and dialyzed against the same buffer. The volume of the Ig fraction was adjusted with phosphate buffer to the initial volume of the serum. ELISA. Direct ELISA was performed on microtiter plates (Nunc) coated with purified peptides (40 ng of antigen per well in 0.1 ml sodium carbonate buffer, 0.1 M, pH 9.6) for 2 hrs at 37” or one night at 5”. After three washings in 0.02 M PBS, pH 7.4 (containing 0.1% Tween 20 and 0.02% sodium azide), the plates were incubated for 1.5 hr at 37” with guinea pig antibodies diluted 1:500 or I:300 in PBS buffer containing 1% normal goat serum. After three washings, a new incubation performed (1.5 hr at 37”) with an anti-
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guinea pig IgG alkaline phosphatase conjugate developed in goat (Sigma A-7664), diluted 1:lOOO. Finally, the enzymatic activity was assessed by the addition of the substrate (1 mg/ml p-nitrophenyl phosphate disodium in 0.05 M carbonate buffer, pH 9.8). The reaction was not stopped and the optical density was determined every 10 min at 410 nm using a Dynatech ELISA reader. Absorption of antibodies. Increasing amounts of peptides, from 0 to 25 CLp,were each redissolved in 15 ul of antibodies diluted with 45 u1 of PBS (50 mM, pH 7.2). The tubes were incubated 1 hr at 37” and one night at 5”, and centrifuged at 20,OOOgfor 20 min. The supematants were diluted to obtain a final dilution of l/300 or l/500 for all samples. The ELISA was carried out as before.
RESULTS Immunoreactivity of the Antibodies against the Major Lobster Sinus Gland Peptides Figure 1 shows a typical elution pattern resulting from the fractionation of hot acetic acid sinus gland extract in the analytical propanol/water/TFA HPLC system. The major peptides, eluted between 24 and 26% of n-propanol, are numbered 1 to 6 according to their elution positions. The shoulder between 3 and 4 (numbered 3’) appears consistently. The amount extractable from one SG was estimated as 250 ng for peptide I,80 ng for peptide 2,55 ng for peptide 3,95 ng for peptide 4, 260 ng for peptide 5, and 210 ng for peptide 6.
SOYEZ
After extraction in cold acetic acid, two extra peaks (numbered 7 and 8) with almost the same retention times as peaks 1 and 2 were present, in addition to more polar peptides eluted in earlier zone of the gradient (Fig. 2). A lengthy extraction (1.5 to 2 hr) of SG homogenate in cold acid often results in the disappearance of peaks 1, 2, 5, and 6. Peaks 3, 3’, and 4 are not modified (chromatogram not shown). Peaks 7, 8, and 3’, as well as the major peptides 1 to 6, have been included in the immunological studies reported below. Determination of the immunoreactivity of anti-3 and anti-4 antibodies by ELBA shows a positive reaction with all peptides tested (Table 1). Maximal reactions are obtained with peptides 3’ and 4, then with 3. Anti-l and anti-5 antibodies do not recognize peptides 3, 3’, and 4 and display the strongest responses with peptides 1 and 2 (Table 1). As mentioned on Table 1, some variations of the reactivity are observed from one experiment to another-and from one peptide preparation to anotherwithout modifications of the general features of the responses. The results described above indicate that anti-3 and anti-4 antibodies display a poor specificity toward the different SG peptides. In order to investigate whether this observation resulted from the presence of
I..% propanol
115
t (min) FIG. 1. RP-HPLC elution profile of lobster sinus glands extracted by hot acetic acid. Thirty sinus glands were extracted during 5 min in 10% acetic acid at 85”. Column, Nucleosil C18, 5 urn (25 cm x 0.46 cm i.d.); solvents, 0.1% TFA in water and 0.1% TFA in n-propanol. Flow rate, 0.75 ml/min. The gradient of n-propanol is indicated by the dotted line. AUFS, absorbance unit full scale; t, time of elution in minutes. The major peaks are numbered I to 6. Peak 3 was characterized as vitellogenesis inhibitor (VIH) in previous studies (see the introduction). 0
10
20
30
40
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.....% propanol 40 6
AUFS 0,002 I .-’
,_._._._._._.__... .............. .:’ ,.I’ ._.’ ,..’ 1” ..’ _____._. II:.’...’
J
20
10
0
30
40
I’
30
:
20
t (mn)
FIG. 2. RP-HPLC profile of lobster sinus glands extracted by cold acetic acid. Forty sinus glands were extracted in 10% acetic acid at room temperature during 30 min. Column, Nucleosil C18, 5 pm (25 cm x 0.46 cm i.d.); solvents, 0.1% TFA in water and 0.1% TFA in n-propanol. Flow rate, 0.75 mk’min. The gradient of n-propanol is indicated by the dotted line. AUF& absorbance unit full scale; t, time of elution in minutes. The main difference with the chromatogram presented in Fig. 1 is the presence of peaks 7 and 8, eluted around 30 min.
several types of IgG in the antibodies preparations or from the existence of common epitopes on the different peptides, absorptions of these antibodies by the reacting peptides were realized, followed by ELISA determination of the remaining immunoreactivity. Absorption of Anti-3 Antibodies Peptide 5 and Peptide 7
by
When 15 J.L~of anti-3 antibodies
are abTABLE
IMMUNOREACTIVITY
sorbed by increasing amounts of peptide 5, the reactivity of the remaining immunoglobulins with peptides 3, 3’, and 4 is not affected (Fig. 3). In contrast, absorption with 250 ng of peptide 5 is sufficient to decrease at least fivefold the reaction with peptides 1, 2, 5, 6, 7, and 8. With peptides 1 and 2 only, a reaction is still present after absorption with 5 kg of peptide 5. Similar results are obtained when anti-3 antibodies are absorbed by peptide 7 (Fig. 4). Absorptions of anti-3 antibodies by 1
OF ANTIBODIES RAISED AGAINST FOUR PEPTIDES FROM Homarus americanus SINUS GLANDS
PURIFIED
BY
HPLC
Antibody Peptide
Anti-l
Anti-3
Anti-4
Anti-5
1
+ +I+ + +
2 3 3' 4 5 6 7 8
+++ 0 0 0 + + +I-?- -t -k-k
++
++
++ + +I+ + + -I- +I-!- + + + + +I-+ + + + +I+ + + ++ ++
++ + -k/i- + + + +I+ + + + + +I+ + + + + + + +
++++ ++++ 0 0 0 + +I+ + + + ++I+++ +++ +++
Note. The antibodies were tested in ELISA against the homologous antigens (bold figures) and the other major sinus gland neuropeptides. The relative immunoreactivity is expressed from 0 to + + + + .
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SOYEZ 1.5 -
E
1.0.
z 2
0.5
01
2
3
3’ 4 PEPTIDE
5
6
7
1
8
FIG. 3. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 5, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 t.~l of undiluted antibodies. Optical density values correspond to 30-min readings.
higher amounts of peptide 5 were carried out in order to determine if the immunoreactivity with peptide 1 (or 2) could be completely suppressed. As shown in Fig. 5, this reactivity remains even when 25 pg of peptide are used for absorbing 15 p,l of antibodies solution. In these conditions, the reactivity with peptide 3 is not significantly reduced . Absorption of Anti-3 and Anti-4 Antibodies by Peptide 2 Figure 6 shows that a small amount of peptide 2 (250 ng/lS p,l antibodies solution)
3
5
7
PEPTIDE
FIG. 5. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with large quantities of peptide 5, as revealed by ELISA against peptides 1,3,5, and 7. In the insert: amount of peptide added to 15 l.d of undiluted antibodies. Optical density values correspond to 20-min readings.
suppresses the reaction of anti-3 antibodies with peptides 1 and 2, while the reactivity for peptides 5,6, 7, and 8 is decreased by a twofold factor. Increased amounts of antigen do not significantly modify the response. No significant modification of the response with peptides 3, 3’, and 4 is observed in this experiment. Similar features are observed when the ELISA is realized with anti-4 antibodies (Fig. 7), though the reactivity of these antibodies with 1 and 2 peptides cannot be totally suppressed. Absorption of Anti-3 Antibodies Peptides 2 + 5 When the anti-3 antibodies
by are simulta-
Is-
E E
1.0-
z 2
0.5.
3
3’
4 PEPTI
5
6
7
8
DE
FIG. 4. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 7, as revealed by ELBA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 pl of undiluted antibodies. Optical density values correspond to 30-min readings.
1
2
3
3’
4
5
6
7
8
PEPTIDE
FIG. 6. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 2, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ~1 of undiluted antibodies. Optical density values correspond to 20-min readings.
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NEUROHORMONES I.00
0.80
.
E c
Peptlde
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I
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5
.
Peptide
1
0.60
s
;
I
=
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.
.
, 0
.
.
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0.20
2
3
3’
4
s
6
8
7
PEPTIDE
7. Immunoreactivity of anti-4 antibodies witbout absorption and after absorption with increasing quantities of peptide 2, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ul of undiluted antibodies. Optical density values correspond to 20min readings. FIG.
neously absorbed by equal amounts of peptides 2 and 5, the response with peptides 1, 2, 5, 6, 7, and 8 is completely abolished (Fig. 8). The reaction with 3, 3’, and 4 is unaffected, whatever be the quantity of 2 + 5 peptides added. Absorption of Anti-3 Antibodies Peptide 4
by
Immunoreactivity of anti-3 antibodies has been investigated after absorption with increasing amounts of peptide 4 (Fig. 9). A
0 1
2
3
3’
4
5
6
7
8
PEPTIDE
FIG. 8. Immunoreactivity of anti-3 (anti-VIH) antibodies without absorption and after absorption with increasing quantities of peptide 2 + peptide 5, as revealed by ELISA against the major peptides of the lobster sinus gland. In the insert: amount of peptide added to 15 ul of undiluted antibodies. Optical density values correspond to 25min readings.
0
10’ 10’ 10’ ABSORPTION (ng of peptide)
10’
FIG. 9. ELBA absorption profiles of anti-3 (antiVIH) antibodies absorbed by increasing amounts of peptide 4 and tested against peptides 1, 3, 5, and 7. Abscissa represents the amount of peptide 4 used for absorption on a logarithmic scale. Optical density values correspond to 20-min readings.
sigmoidal absorption curve is obtained when the antibodies are tested against peptide 3. The reaction is completely abolished when 15 l.rl of anti-3 antibodies are absorbed with 5 and 10 p.g of peptide 4. In contrast, the reactivity against peptides 5 and 7 is not modified whereas a slight reduction of the response is observed with peptide 1 when 5 or 10 p,g of antigen 4 is added to the antibodies. DISCUSSION
The present investigation on the specificity of guinea pig antisera raised against purified lobster neuropeptides indicates the existence, in these molecules, of some common immunological characteristics. ELISA experiments realized with antibodies raised against peptide 1 and against peptide 5 indicates that peptides 1, 2, 5, 6, 7, and 8 are recognized by both antibodies and therefore do possess common antigenic determinants. On the other hand, peptides 3 and 4 (as well as 3’) do not react with the anti-l and anti-5 antibodies and are highly recognized by anti-3 and anti-4 antibodies (peptide 4 being generally more reactive). As a matter of fact, all the nine peptides tested (i.e., 1, 2, 3, 3’, 4, 5, 6, 7, and 8) are recognized by anti-3 (or anti-4) antibodies.
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After selective absorptions, however, the anti-3 antibodies become strictly specific for peptides 3, 3’, and 4. Therefore, these peptides seem closely related. This result reinforces and completes previous work which indicates that peptide 3, biologically characterized as VIH (Soyez et al., 1987), and peptide 4 may be structurally related since they display analogous amino acid composition and pHi. (Soyez et al., 1988). In a previous study, antisera were raised in Biozzi mice against peak 3. Two out of three mouse antisera reacted slightly with peak 1 peptide. After absorption with this peptide the antisera recognized only peak 3. In no case did the mouse antisera crossreact with peptide 4 (Meusy ef al., 1987). The difference with the results exposed in the present report could be ascribed to the immunization itself which was very light in the experiment with mice: 7.7 kg of peptide per mouse in three injections as compared to 55 pg per guinea pig in five injections for the present study. The absorption of anti-3 antibodies by peptide 5 (or 7) completely abolished the reaction with peptides 5, 6, 7, and 8. Reactivity with peptides 1 and 2 strongly decreased when increasing amounts of peptide 5 were used, but could not be completely suppressed, while reaction with peptides 3, 3’) and 4 was not affected. This result is very surprising since the antibodies were not supposed to be raised against any other epitopes than those of the injected peptide 3. A likely hypothesis could be that the epitopes involved in the absorptions are not present-or not reactant-in those three last peptides. A trivial error during preparation or injections is highly improbable since strict precautions have been taken; furthermore, it must be recalled that antibodies against peptides 1 and 5 did not react with peptides 3, 3’ and 4 (it would not be the case if some animals had been mixed). It is likely that an epitope of peptide 3, also present in peptides 5 and 7, was immunogenic upon injections in guinea pigs
SOYEZ
but was not antigenic in ELISA determinations or during absorption processes. This epitope could be masked in native peptide 3 (or 4) and would be accessible during immunization. Such a hypothesis agrees with recent immunological theories where the cleavage of antigens within endosomes of B-lymphocytes is described as an early step of immunization process, followed by the raising of antibodies against some peptidic fragments (review in Long, 1989). The existence of a common epitope present in the major sinus gland peptides and masked in native peptides 3 or 4 should be proved by demonstrating the existence of structural homologies between all these peptides. The absorption of anti-3 and anti-4 peptides by peptide 2 gave results comparable to those obtained after absorptions with peptides 5 or 7: the reactivity with peptides 1 and 2 was suppressed, the response with peptides 5, 6, 7, and 8 was only decreased down to some steady level, while reactivity with peptides 3, 3’, and 4 was not changed. So, we can postulate that the epitopes of peptides 1 and 2 are completely different from the reactant epitopes of peptides 3,3’, and 4 and are partially common with those of peptides 5, 6, 7, and 8. This agrees with recent data which indicates that the four peptides 1, 2, 5, and 6 display a hyperglycemic activity though the first two peptides present a different isoelectric point, molecular mass, and amino acid composition from the last two (Soyez et al., 1990). As expected, after absorptions of anti-3 antibodies by 2 + 5 peptides, only peptides 3, 3’, and 4 are recognized. After absorption of anti-3 antibodies by peptide 4, their reaction with peptide 3 falls down to zero, following a standard sigmoidal curve. This result confirms that the reacting epitopes of peptides 3 and 4 are identical, while those of peptides 1, 5, and 7 seem different from them. When repeating ELISA experiments, quantitative variations of reactivity were often observed. Since aliquots of the same
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NEUROHORMONES
antibody preparations were used each time, this feature may be attributed to the antigens themselves. Small inaccuracies in quantification and solubilization can contribute to these variations by physicochemical modification(s) during storage, such as oxidation of methionine residues for example, can also be involved. We frequently observed that frozen and thawed antigens are more reactive than those freshly prepared. This could be related to the existence of some masked epitope in native peptides, as discussed above. Peptides 7 and 8 were observed only after extraction of SG in acetic acid in cold or at room temperature, and their presence is correlated with a reduction of the amount of peptides 5 and 6. This observation agrees with the results of Chang et al. (1987): these authors characterized a doublet peak with molt-inhibiting activity (peak 8) from the SG of the lobster H. americanus. These peptides slowly degrade in cold acetic acid, generating shorter peptides (doublet peak 5) which lack bioactivity. On the basis of chromatographic behaviour and amino acid composition analyses, Soyez et al. (1990) suggest that hyperglycemic peptides 5 + 6 are homologous to doublet peak 8 from Chang et al. (1987). Therefore doublet peak 5 described by these last authors could correspond to peptides 7 + 8 studied in the present report. Close structural relationships between peptides 5 + 6 and 7 + 8 are further confirmed by the identical immunological behaviour of all these peptides in the absorption experiments of anti-3 antibodies. As mentioned above, our experiments indicate that peptides 1, 2, 5, 6, 7, and 8 display at least one identical epitope. Furthermore, CHH peptides (as well as VIH) (Meusy et al., 1987) are not strictly species specific. Previous results obtained by dot immunobinding assay (DIA) or ELISA suggest that hyperglycemic peptides from other crustacean species may display the same epitope: an antisera raised against
CHH from the crayfish A. leptodactylus’ recognized the lobster CHH related peptides (Van Deijnen, 1986; Tensen et al., 1989 and Meusy, unpublished results). The same observation was made with an antisera raised against CHH from Orconectes limosus* (Meusy, unpublished results). Therefore structural analogies may exist between hyperglycemia inducing peptides within, at least, the astacidea. To conclude, among the HPLC-purified peptides from the lobster sinus gland, three immunological families can be distinguished: the peptides 1, 2 group (CHH group number 1); the peptides 5, 6, 7, 8 group (CHH group number 2); and the peptides 3, 3’, 4 group (VIH group). These groups do present some identical antigenic determinant(s) and it will now be interesting to elucidate the structural relationships between these peptides in relation to their synthesis pathway and physiological significance. ACKNOWLEDGMENTS The authors thank Dr. Gerard Conan and Ms. Claire Doucette for having supervised the supply of lobster eyestalks and Mr. Moret and the staff of CNRZ-INRA for their assistance during the immunization procedures. We are grateful to Ms. Madeleine Martin for her technical collaboration and to our colleagues for their helpful participation in the preparation of the sinus glands. We thank Professor J. Charlemagne for his critical reading of our manuscript.
REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Chang, E. S., Bruce, M. J., and Newcomb, R. W. (1987). Purification and amino acid composition of a peptide with molt-inhibiting activity from the lobster, Homarus americanus. Gen. Comp. Endocrinol. 65, 56-64.
’ From Dr. F. Van Herp. ’ From Professor R. Keller.
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Kallen, J., and Meusy, J.-J. (1989). Do the neurohormanes VIH (Vitellogenesis Inhibiting Hormone) and CHH (Crustacean Hyperglycemic Hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster, Homarus americanus. Invertebr. Reprod. Dev. 16, 43-52. Keller, R. (1969). Untersuchungen zur artspezititlt eines crustaceenhormons. 2. Vgl. Physiol. 63, 137-14s. Keller, R. (1977). Comparative electrophoretic studies of crustacean neurosecretory hyperglycemic and melanophore-stimulating hormones from isolated sinus glands. J. Comp. Physiol. 122, 359-373. Kleinholz, L. H., and Keller, R. (1979). Endocrine regulation in Crustacea. In “Hormones and Evolution” (E. J. W. Barrington, Ed.), Vol. 1, pp. 159-213. Academic Press, New York. Leuven R. S. E. W., Jaros, P. P., Van Herp, F., and Keller, R. (1982). Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. Gen. Camp. Endocrinol. 46, 288-296. Long, E. 0. (1989). Intracellular traftic and antigen processing. Immunol. Today 10, 232-234. Meusy, J.-J., Martin, G., Soyez, D., Van Deijnen, J. E., and Gallo, J.-M. (1987). Immunochemical and immunocytochemical studies of the crustacean vitellogenesis-inhibiting hormone. Gen. Comp. Endocrinol. 55, 208-216.
Meusy, J.-J., and Payen, G. G. (1988). Female reproduction in Malacostracan crustacea. Zool. Sci. 5, 217-265. Soyez, D., Noel, P. Y., Van Deijnen, J. E., Martin, M., Morel, A., and Payen, G. G. (1990). Neuropeptides from the sinus gland of the lobster Homarus americanus. Characterization of hyperglycemic peptides. Gen. Comp. Endocrinol. 79, 261-274. Soyez, D., Van De&ten, J. E., Laverdure, A. M., Martin, M., Meusy, J.-J., Noel, P. Y., Payen, G. G., Tensen, C. P., and Van Herp, F. (1988). Neuropeptides from the sinus gland of the lobster Homarus americanus. Gen. Comp. Endocrinol. 74, 319 [Abstract]. Soyez, D., Van Deijnen, J. E., and Martin, M. (1987). Isolation and characterization of a vitellogenesisinhibiting factor from sinus glands of the lobster, Homarus americanus. J. Exp. Zoo/. 244,479-484. Tensen, C. P., Janssen, K. P. C., and Van Herp, F. (1989). Isolation, characterization and physiological specificity of the crustacean hyperglycemic factors from the sinus glands of the lobster, Homarus americanus (Milne-Edwards). Invertebr. Reprod. Dev. 16, 155-164. Van Deijnen, J. E. (1986). ‘Structural and Biochemical Investigations into the Neuroendocrine System of the Optic Ganglia of Decapod Crustacea.” Ph.D. Thesis, Catholic University, Nijmegen, The Netherlands.
