Tài liệu Báo cáo khoa học: Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family docx

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Tài liệu Báo cáo khoa học: Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family docx

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Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family Ce ´ line Ollivaux 1,2,3 , Dominique Gallois 4 , Mohamed Amiche 4 , Maryse Boscame ´ ric 4 and Daniel Soyez 4 1 Universite ´ Pierre et Marie Curie – Paris 06, UMR 7150 Mer et Sante ´ ,E ´ quipe Physiologie Compare ´ e des Erythrocytes, Station Biologique de Roscoff, France 2 Centre National de la Recherche Scientifique, UMR 7150, Station Biologique de Roscoff, France 3 Universite ´ Europe ´ enne de Bretagne, UEB, Rennes, France 4 Equipe Biogene ` se des Signaux Peptidiques, ER3, Universite ´ Paris, France Introduction Modification of the chirality of a single aminoacyl resi- due within a peptide chain is a subtle and intriguing mechanism that remains poorly known to date, and which leads to structural and functional diversification of eukaryotic bioactive peptides. Subsequent to the study by Montecucchi et al. [1] describing the presence of a d-alanyl residue at the second position of dermor- phin, a powerful opioid peptide isolated from skin secretions of the tree frog Phyllomedusa sauvagei, such a phenomenon has been reported in vertebrates, in different opioid peptides [2], in antibacterial and haemolytic peptides from frog skin [3], as well as in venom from the mammal Platypus [4] (Table 1). In invertebrates, d-amino acid containing peptides (DAACPs) were isolated from the nervous system of molluscs and crustaceans [5], and from venom of a Keywords aminoacyl isomerization; confocal laser scanning microscopy; crustacean hyperglycaemic hormone; MALDI-TOF MS; vitellogenesis inhibiting hormone Correspondence C. Ollivaux, Universite ´ Pierre et Marie Curie – Paris 06, UMR 7150 Mer et Sante ´ ,E ´ quipe Physiologie Compare ´ e des Erythrocytes, Station Biologique de Roscoff, 29682 Roscoff, Cedex, France Fax: +33 1 44 27 23 61 Tel.: +33 1 44 27 22 62 E-mail: celine.ollivaux@upmc.fr (Received 29 March 2009, revised 23 June 2009, accepted 26 June 2009) doi:10.1111/j.1742-4658.2009.07180.x d-aminoacyl residues have been detected in various animal peptides from several taxa, especially vertebrates and arthropods. This unusual polymor- phism was shown to occur in isoforms of the crustacean hyperglycaemic hormone (CHH) of the American lobster because a d-phenylalanyl residue was found in position 3 of the sequence (CHH and d-Phe3 CHH). In the present study, we report the detailed strategy used to characterize, in the lobster neuroendocrine system, isomers of another member of the CHH family, vitellogenesis inhibiting hormone (VIH). We have demonstrated that the fourth residue is either an l-orad- tryptophanyl residue (VIH and d-Trp4 VIH). Furthermore, use of antisera specifically recognizing the epimers of CHH and VIH reveals that aminoacyl isomerization occurs in specialized cells of the X organ–sinus gland neurosecretory system and that the d-forms of the two neuropeptides are not only present in the same cells, but, importantly, also are co-packaged within the same secretory vesicles. Abbreviations CHH, crustacean hyperglycaemic hormone; DAACP, D-amino acid containing peptide; gp, guinea pig; PTM, post-translational modification; r, rat; rb, rabbit; VIH, vitellogenesis inhibiting hormone; XO, X organ. 4790 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS spider [6] and cone snails [7]. l-tod-aminoacyl isomerization, or epimerization, comprises a true post- translational modification (PTM) of peptides [8], as are glycosylation or phosphorylation, where an l-residue, which is always the same in a given peptide, is con- verted into its d-counterpart [9]. This PTM occurs at a late step of peptide precursor processing, after the cleavage of the propeptide [10]. The nature of the d-res- idue varies according to the peptides, although it has consistently been found at the extremities of the sequence at the second or third position, mostly at the N-terminus and more rarely at the C-terminal end, except in several conotoxins (e.g. the contryphan, an octapeptide with a d-tryptophan in the fourth position) [11] (Table 1). This intriguing modification is likely to involve a specific enzyme, designated as peptidylaminoa- cyl-l ⁄ d-isomerase or, more simply, isomerase [12] or epimerase [13]. Amino acid sequences of putative enzymes have been obtained from venom of the spider Agenelopsis aperta [14], and more recently from the skin secretion of Bombina frogs [15]. Unexpectedly, these enzymes appear to be totally unrelated with regard to their structures and their target residues. In addition, such an isomerase has been isolated from platypus venom, but remains unsequenced [12,16]. Although d-amino acids have been mostly found in the sequence of small (i.e. 3–40 residues) peptides, remarkable exceptions include peptides belonging to the crustacean hyperglycaemic hormone (CHH) family, which are 72–83 residues long, elaborated in the major crustacean neurosecretory system, the X organ–sinus gland complex. In several crustacean species, two epimers of CHH can be purified, which differ in the configuration of the phenylalanyl residue at position 3 (i.e. either l or d). To date, the presence of both CHHs (named CHH and d-Phe3 CHH) has been dem- onstrated only in Astacoidea (crayfish and lobsters), where CHH displays the same N-terminal aminoacyl sequence, at least up to the tenth residue [17]. Phe3 isomerization has major physiological consequences on CHH biological activity because the all-l-peptide is strictly hyperglycaemic, whereas d-Phe3 CHH may exhibit, in addition to higher hyperglycaemic potency, other functions, such as moult inhibition [18] or osmo- regulation [19]. At the present, it is unclear whether functional differences between CHH isomers are related to binding to specific receptors or to differences in haemolymphatic clearance rate. Indeed, DAACPs are known to be more stable because they are less sus- ceptible to protease degradation. As noted above, CHH constitutes the archetype of an original peptide family mostly found in crustaceans, the CHH family, which contains other essential neurohormones, such as moult-inhibiting hormone, mandibular organ inhibiting hormone and vitellogenesis inhibiting hormone (VIH; also called gonad inhibiting hormone) [20]. In Homarus americanus, where d-Phe3 CHHs were first identified [21], VIH is present in the neurohaemal organs, the sinus glands, as two isoforms (VIH I and II) with an identical 77-amino acid sequence, molecu- lar mass (9135 Da) and pI (6.8) [22]. Only one cDNA was cloned, encoding a precursor with a signal peptide directly flanking the progenitor VIH sequence [23]. With regard to its biological functions, VIH may inhi- bit vitellogenesis synthesis in ovary or at extra-ovarian sites and also may inhibit vitellogenin uptake in oocytes [24]. When American lobster VIHs were tested in a heterologous in vivo assay, only VIH I, the hydro- philic form, demonstrated significant inhibitory activity with respect to repressing oocyte growth that had been induced by eyestalk removal in grass shrimps [25]. To date, no function has been assigned to VIH II, the hydrophobic form. VIH has also been detected in male American lobster sinus gland [26], in the Norway lobster Nephrops nor- vegicus [27] and in the woodlouse Armadillidium vulgare [28]. In the latter species, sinus gland grafting experi- ments have suggested that VIH could be involved in androgenic gland growth [29]. In the present study, we describe an experimental strategy that was applied to determine the difference between VIH I and II from the X organ–sinus gland system of the lobster H. americanus. We demonstrate that VIHs differ in the chirality of the tryptophan at position 4. This result has been exploited to develop specific antisera recognizing specifically the N-terminal end of VIH and d -Trp4 VIH, which has allowed Table 1. D-amino acid containing peptides in animals. Bold and underlined letters indicate the D-residues. CHH, crustacean hyper- glycemic hormone; VIH, vitellogenesis inhibiting hormone; OvCNP, Ornithorhyncus venom C-type natriuretic peptide; DPL, defensin- like peptide. Organism Tissue Name Sequence Reference Frog Dermal gland Dermorphin Y AFGTPSNH2 [1] – – Bombinin H I IGPVLG [3] Platypus Venom gland OvCNP L LHDHPN [32] – – DLP I MFFEMQ [4] Snail Ganglia ⁄ heart Achatin G FAD [31] Mussel Muscle FFRF amide A LAGDHFFRFNH2 [52] Aplysia Heart NdWamide N WFNH2 [53] Cone snail Venom duct Contryphan GChP WEPWC [11] Spider Venom gland Xagatoxin MEGL SFA [50] Lobster Sinus gland CHH pQEV FDQAC [21] – – VIH ASA WFTN Present Study C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4791 identification of VIHs from the European lobster Homarus gammarus sinus gland extracts [30]. In such a complex system as this, where l- and d- epimers of different neuropeptides are present in the neuroendocrine organ, a puzzling question remains unanswered: how are these peptides distributed in the different cells of the X organ–sinus gland system? In other words, what is the cellular specificity of the isom- erization process? To answer this question, the distri- bution of VIH and CHH isomers within the lobster X organ–sinus gland complex was studied by immuno- fluorescence ⁄ confocal microscopy. This technique was supplemented by an immunogold-electron microscopy study of the neuronal endings in the sinus gland to determine the subcellular localization of the different epimers. Taken together, these results allow a discus- sion of the existence of enzyme(s) converting l-to d- residues in the peptide chain in terms of substrate specificity and availability. Results Purification and characterization of VIH I and II VIHs were purified from H. americanus sinus gland extract by RP-HPLC. Figure 1 shows a typical elution pattern resulting from the fractionation of an extract of 30 lobster sinus glands. The major peptides were eluted between 38% and 40% acetonitrile, and were identified as CHH B, d-Phe3 CHH B, VIH I, VIH II, CHH A and d-Phe3 CHH A, respectively, according to their elution order, by reference to a previous study [21]. These assumptions were confirmed by a direct ELISA performed on aliquots of the different frac- tions, using antisera anti-4 (recognizing both VIHs) and the two antisera discriminating CHH and d-Phe3 CHH, guinea pig (gp)-anti-pQl and rabbit (rb)-anti-pQd, respectively (not shown). Examination of chromatograms from different sinus gland batches shows a constant abundance ratio of the different pep- tides, with the VIH I peak being half the size of the VIH II peak (Fig. 1). The rationale of our experimental approach for identifying a putative d-residue in VIH was reliant on: (a) DAACPs being generally more hydrophobic than their l-counterparts in most DAACPs from eukaryotes known to date, including CHH, and (b) the d-residue being found near the N-terminal end, between the sec- ond and the fourth position of the sequence. Conse- quently, we hypothesized that the d-residue was present in the N-terminal heptapeptide of VIH II, the hydrophobic form. In a first attempt to identify the putative d-residue in VIH, we considered that, in CHH A and B, the d-amino acid is the phenylalanine at position 3 [21]. We assessed whether the nature of the modified residue (Phe3 in CHH and Phe 5 in VIH) or its position (Phe3 in CHH and Ala3 in VIH) may be conserved in CHH and VIH. Thus, heptapeptides corresponding to the N-terminal sequence of VIH were synthesized with different configurations (i.e. Hep-l, Hep-dA3 and Hep-dF5). Samples of VIHs (ten sinus gland equivalents, i.e.  50 pmol VIH I and  100 pmol VIH II) were cleaved with endoproteinase Asp-N and the resulting fragments were separated by RP-HPLC. After fractionation of VIH I hydrolysate, one fragment was eluted with the same retention time as the synthetic peptide Hep-l (Fig. 2A). The mass of this fragment was determined by MALDI-TOF MS to be 796 Da, which is a value identical to the mass of Hep-l. Because no other predicted fragment from endoproteinase-Asp-N digestion of VIH displayed a similar mass, this fragment could be identified as the N-terminal heptapeptide of VIH I. After similar cleav- age of VIH II by endo-Asp-N and RP-HPLC, MS analysis indicated that a peptide with a molecular mass of 795.9 Da was eluted at 31.5 min (i.e. later than Hep-l, Hep-dA3 and Hep-d F5; not shown). In a next step, to determine which residue may be in the d-configuration in the fragments resulting from VIH II digestion, we considered that numerous DAACPs display a d-residue at position 2 (in snail excitatory peptides [31], in frog opioid peptides [1] and in platypus venom [32]). In addition, the tryptophan residue at position 4 may be a good candidate because contryphan, a conotoxin isolated from gastropod venom has a d-Trp4 [11]. To test these hypotheses, the synthetic peptides Hep-dS2 and Hep-dW4 were used Acetonitrile (%) 39 39. 5 40 0.1 u AU 220 nm CHH B CHH A D-Phe 3 CHH B D-Phe 3 CHH A 49 53 Retention time (min) VIH I VIH II 45 41 Fig. 1. RP-HPLC profile of an acetic acid extract of 30 lobster sinus glands. Only the part of the chromatogram where CHHs and VIHs are eluted is shown. The nature of the ultraviolet absorbance peaks was assessed by ELISA as well as by comparison with previously published similar analyses [26]. Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al. 4792 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS as standards. Figure 2B shows that the elution time of Hep-dW4 is identical to that of the VIH II fragment. By MALDI-TOF MS, the mass of this fragment was found to be 795.8 Da, similar to the synthetic peptide. Moreover, co-injection on RP-HPLC of the VIH II fragment and Hep-dW4 resulted in a single symmetri- cal peak (not shown). Therefore, the results obtained indicated that the two VIH isomers differed by the configuration of the tryptophan at the fourth position of the sequence and, consequently, they were named VIH and d-Trp4 VIH. This assumption was further confirmed by immuno- assays realized with the antisera directed against the N-terminal decapeptides of VIH with all l-residues [rat (r)-anti-l] or with the tryptophan residue in d-configu- ration (gp-anti-dW4). Indeed, ELISA using affinity- purified antisera with the different heptapeptides demonstrated a strict specificity of these antisera for the antigen peptide, with cross-reactivity for the other heptapeptides being lower than 1% (not shown). Accordingly, when ELISA was performed on fraction aliquots from RP-HPLC of sinus gland extract (as in Fig. 1), no signal was obtained using these antisera with fractions corresponding to CHHs A and CHHs B, whereas r-anti-l gave a strong signal with VIH I only and gp-anti-dW4 with VIH II exclusively, which confirmed unambiguously the presence of the d-Trp in VIH II (not shown). Characterization of cell types in relation to CHH and VIH isomers To study the distribution of CHHs and VIHs in the X organ cells, immunohistochemical labelling of whole-mounts of X organ–sinus gland complexes was realized using different sets of antibodies. In addition, immunogold labelling was performed on sinus gland sections to localize these peptides at the subcellular level, within secretory granules. Localization of VIH and D-Trp4 VIH in X organ neuroendocrine cells Confocal analyses of whole-mounts of X organ–sinus gland complexes of the lobster H. americanus were per- formed after double immunofluorescent labelling using purified antisera r-anti-l and gp-anti-dW4. Different cell types were observed: the larger neuroendocrine cell bodies (70 ± 7 lm diameter soma) were strongly labelled with gp-anti-dW4 (green cells; Fig. 3A), with the labelling being cytoplasmic and granular; only some of these cells, with a smaller diameter (56 ± 7 lm) were also stained with r-anti-l, the yel- low ⁄ orange colour, variable in a same organ, attesting to labelling with both antisera. For the sake of clarity, both types are subsequently referred to as d-VIH cells. Smaller VIH-producing cells (31 ± 7 lm diameter soma) were grouped in a distinct region. Their peri- karya were immunoreactive with r-anti-l exclusively (red cells called l-VIH cells). A total of 14 d-VIH cells (nine green and five yellow cell bodies) and 19 l-VIH cells (red soma) were counted per X organ. Most Hep-L Hep-DA3 0.01AU 220 nm Hep-DF5 34 36 Acetonitrile (%) 32 Retention time (min) 26 30 34 22 38 Hep-DW4 36 Hep-L Hep-DF5 Hep- DA3 Hep- DS2 0.01AU 220 nm Acetonitrile (%) 32 34 Retention time (min) 26 30 34 22 38 A B Fig. 2. (A) RP-HPLC profile of VIH I digest (ten sinus gland equiva- lents). Only the part of the chromatogram where fragments elute is shown. The nature of the ultraviolet absorbance peaks was assessed by comparison with retention times of standards (arrows) (i.e. heptapeptides Hep- L, Hep-DA3 and Hep-DF5) coupled with MALDI-TOF mass analysis. (B) RP-HPLC profile of VIH II digest (ten sinus gland equivalents). Compared with the previous analysis shown in Fig. 2A, the synthetic heptapeptides Hep- DS2 and Hep- DW4 were added to the standard mixture. C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4793 C 50 µm A 50 µm B 50 µm R LG SG XO ME MI MT H 20 µm D 20 µm E 50 µm F 20 µm G 50 µm Fig. 3. Confocal micrographs of double immunolabelled whole mounts of lobster X organ–sinus gland. Images were collected as a focal series and processed to create 2D projections (single composite images). Central drawing: schematic representation of the lobster eyestalk nervous structures and the neuroendocrine complex (X organ–sinus gland). R, retina; LG, lamina ganglionaris; ME, medulla externa; MI, medulla interna; MT, medulla terminalis. (A) General view of X organ labelled with r-anti- L and gp-anti-DW4 to visualize small L-VIH cells (red) and larger D-VIH cells (green or yellow). (B) Axonal arborizations of both cell types observed in the sinus gland (red for L-VIH varicosities and green for D-VIH ones). (C) Distribution of CHH cells in X organ showing green cell bodies (L-CHH cells) labelled only with gp-anti-pQL and orange somata ( D-CHH cells) corresponding to labelling with gp-anti-pQL and rb-anti-pQD antisera. (D) Enlargement of axon terminals in the sinus gland showing both types of secretory granules corresponding to L-CHH cells (green) and D-CHH cells (red). (E) Immunolocalization of D-Trp4 VIH and D-Phe3 CHH in the X organ where three cell types were observed : D-CHH cells (red), D-VIH cells (green) and D-cells produc- ing both D-isomers (orange). (F) Enlarged view of the three cell types showing the variations of coloration for D-cells: D-CHH cells (red, thin arrow), D-VIH cells (green, short arrow) and D-cells producing both D-isomers (orange, long arrows). (G) Sinus gland axonal arborizations containing D-Trp4 VIH (green) and D-Phe3 CHH (red). (H) Enlargement of axon terminals in the sinus gland with labelling as in (G). Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al. 4794 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS d- and l-VIH cells appeared segregated, whereas several somas of the two types were dispersed in the X organ (Fig. 3A), most likely as a result of arte- factual displacement of the cell bodies during the prep- aration of the organ. In the sinus gland, different types of axonal arborizations containing VIH, d-Trp4 VIH, or very rarely both, were observed (Fig. 3B). Ultrastructural observations after immunogold label- ling of sinus gland sections reveal the presence of sev- eral types of terminals differing by the morphology, size and electron density of the secretory granules. After double labelling with r-anti-l and gp-anti-dW4, particles were observed on two categories of axon terminals: those exclusively labelled with r-anti-l (l-VIH terminals; Fig. 4A) and a few d-VIH terminals labelled with gp-anti-dW4 (Fig. 4B). No terminals with double labelling were found. In each terminal, the secretory vesicles were densely packed and, although scarce, the gold labelling was strictly restricted to them. Localization of CHH and D-Phe3 CHH in X organ neuroendocrine cells Whole-mounts of H. americanus eyestalks were incubated with antisera gp-anti-pQl and rb-anti-pQ d, recognizing CHH and d-Phe3 CHH, respectively. Confocal micrographs revealed two distinct cell types: either green, labelled only with gp-anti-pQl (l-CHH cells) or orange ⁄ red, labelled with both gp-anti-pQl and rb-anti-pQd antisera in variable proportions, (d-CHH cells; Fig. 3C). In a cluster of 34 cells, 13 d-CHH cells and 21 l-CHH cells were localized in dis- tinct regions of the X organ. The diameter of their cell bodies was 64 ± 8 lm and 44 ± 7 lm, respectively. Intensively stained axons could be traced to the sinus gland where two types of axonal arborizations were observed, either green or red. Rarely, orange colour- ation attested the presence of both CHH types (Fig. 3D). Accordingly, when sinus gland ultrathin sections were observed after double labelling by gp-anti-pQl and rb-anti-pQd, two different categories of terminals were revealed: l-CHH terminals, labelled with gp-anti-pQl (Fig. 4C) and d-CHH terminals with a strong rb-anti- pQd labelling (Fig. 4D). Those terminals were also rarely labelled with gp-anti-pQl (Fig. 4E). Distribution of D-Trp4 VIH and D-Phe3 CHH in X organ neuroendocrine cells To address the question whether d-epimers of CHH and VIH are expressed in the same cells or not, confocal analysis of whole-mounts of X organ–sinus gland complexes was performed after double immunofluo- rescent labelling using specific antisera gp-anti-dW4 and rb-anti-pQd, recognizing d-Trp4 VIH and d-Phe3 CHH, respectively. Three types of cells could be distinguished: seven green cells strongly labelled with the gp-anti-dW4 (56 ± 7 lm diameter soma; pre- viously called d-VIH cells), ten red cells immunoreac- tive with rb-anti-pQd antiserum (60 ± 5 lm diameter soma; previously called d-CHH cells) and five yel- low ⁄ orange cells stained with both antisera, simply called d-cells (65 ± 7 lm diameter soma; Fig. 3E). Among these latter cells, large differences in colour- ation were observed as a result of variations in the relative amounts of both d-isomers (Fig. 3F). Immunohistochemical staining of axonal arborization in the neurohemal organ showed the three cell types with clustered granules immunoreactive either for one antiserum or for both antisera (Fig. 3G,H). To test the hypothesis of vesicular co-packaging of d-epimers of CHH and VIH, double immunogold labelling with various associations of antibodies against the different forms was performed on ultrathin sections for examination by electron microscopy. Using rb-anti-pQd and gp-anti-dW4, specific d-VIH and d-CHH terminals were observed (Fig. 4F–I). Mixed terminals were also detected in other parts of the sinus gland, demonstrating that d-Trp4 VIH and d-Phe3 CHH were not only colocalized in the same terminals (Fig. 4J), but also in same secretory vesicles (Fig. 4K). These three categories of terminals were usually found in different regions of the sinus gland, as described above, but close juxtapositions of differ- ent terminals were sometimes observed (Fig. 4G). Discussion Although the existence of two VIH isoforms with iden- tical sequence, molecular mass and isoelectric point has been known for more than 15 years [22], the nat- ure of the difference between the two peptides had not yet been elucidated. The demonstration of the presence of a d-Phe3 residue in CHH A and B from the H. americanus some years ago [21] opened the possibil- ity that a d-residue may be present in one of the VIH isoforms as well. Furthermore, in a previous study, ELISA experiments using specific antibodies have suggested the presence of a d-residue in VIH from H. gammarus [30]. In the present study, we have demonstrated, using a combination of RP-HPLC, MALDI-TOF MS (peptide mapping) and immunoassays, that the most hydropho- bic VIH isoform contains a d-tryptophanyl residue at position 4, whereas a l-Trp4 is present in the C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4795 500 nm200 nm 200 nm 500 nm500 nm 100 nm 500 nm 200 nm 200 nm 200 nm 500 nm C A B F D GH I K J E Fig. 4. Sections of double immunogold labelling of axon terminals in the lobster sinus gland. (A, B) Double labelling with r-anti-L (10 nm gold particles) and gp-anti- DW4 (20 nm gold particles) antisera. (A) Axon terminal containing secretion granules with only VIH. (B) Axon terminal containing secretion with D-Trp4 VIH. (C–E) Double labelling with gp-anti-pQL (20 nm gold particles) and rb-anti-pQD (10 nm gold particles) antisera. (C) Two neighbouring CHH axon terminals. (D) Axon terminal with only D-Phe3 CHH. (E) Axon terminal showing some secretion granules labelled with both antisera. (F–K) Double labelling with rb-anti-pQ D (10 nm gold particles) and gp-anti-DW4 (20 nm gold particles). (F) Axon terminal containing D-Trp4 VIH. (G) Two neighbouring axon terminals, one containing D-Trp4 VIH and the other D-Phe3 CHH. (H, I) Higher magnifications of D-Trp4 VIH and D-Phe3 CHH containing granules, respectively. (J) General view of an axon terminal containing both D-isomers. (K) Enlarged view of an ending with secretion granules labelled with both rb-anti- pQD and gp-anti-DW4 antisera. Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al. 4796 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS hydrophilic form. The results obtained therefore estab- lish that the lobster neuroendocrine system elaborates a mixture of epimers of two different neurohormones: CHH, d-Phe3 CHH, VIH and d-Trp4 VIH. The coex- istence, in a same organism, of several peptides with a d-residue of different natures and positions has been documented in venoms of cone snails [33] and of platy- pus [32], and also in frog skin secretions [34]. Neverthe- less, no general consensus for the isomerization site, nor for the nature of the surrounding residues, could be demonstrated to date, with the prediction of single d-amino acid presence being relevant exclusively within a well-defined and restricted peptide family, as illus- trated by a recent study of the I 1 -conotoxin superfamily [35]. In the case of the CHH family, the occurrence of DAACPs appears to be problematic to predict because CHH variants with a d-residue have been detected only in Astacidea (rocky lobsters and crayfish), where the N-terminal sequence is pGlu-Val-Phe-Asp-Glu-Ala (with the d-residue always being the Phe3), and not in CHH from other species with close overall sequence similarity, such as the peneid shrimp Penaeus vannamei with the sequence Ser-Leu-Phe-Asp-Pro-Ser . [36], nor even in those species with an identical sequence of four residues, such as the spiny lobster Jasus lalandii: Ala-Val-Phe-Asp-Glu-Ser [37]. However, it is worth noting that the presence of a d-amino acid in CHHs from these last species may be overlooked because no specific investigation procedure, such as amino acid chiral analysis, was ever performed. Even if the presence of DAACPs begins to be well documented in various animal groups, this phenome- non has been rarely studied at the cellular level. Indeed, cellular aspects have been investigated in the serous dermal gland of frogs. However, owing to the peculiar syncytial structure of this organ, the localiza- tion of dermorphin and its all-l counterpart in the dif- ferent cell compartments was difficult to analyze [34]. By contrast, in crustaceans, DAACPs are elaborated in discrete, well-identified neuroendocrine cells located in an easily accessible organ, with these characteristics having already allowed detailed studies of their bio- genesis [10,38–41]. In previous studies, the expression of CHH and VIH in H. americanus X organ–sinus gland complex was investigated at the peptide level, but without distinc- tion of epimers [42]. It was observed by immuno- histochemistry that some cells were labelled only by anti-CHH or anti-VIH antisera, whereas several others were reactive with both antisera, as an indication of the presence of both hormones in these cells. The existence of mixed CHH ⁄ VIH cells was later confirmed at the mRNA level [26]. When, at an early stage of our study, we considered the cellular distribution of CHH and VIH isomers in the lobster X organ, one attractive simple hypothesis was that the mixed (double labelled) cells observed previously could con- stitute the synthesis site of the d-epimer of the two hormones (d-Phe3 CHH and d-Trp4 VIH), whereas the cells labelled exclusively by anti-CHH or by anti- VIH would produce only the l-counterpart of each peptide. To test this initial hypothesis, the organization of CHH- and VIH- expressing cells, in relation to iso- mers, was investigated by immunohistochemistry and immunocytochemistry, using antisera specific to each epimer. Confocal examination of in toto preparations has shown that approximately 33 cells were immunostained with gp-anti-dW4 or r-anti-l antiserum, which agrees with the results obtained in a previously study [26], but diverges from those of other studies [42,43] report- ing a smaller figure (approximately 20 cells). It may be that the number of cells expressing VIH at a given time varies according to the reproductive stage of the animals, such as the haemolymphatic VIH level [44]. Regarding the CHH-producing cells, our observa- tions indicate that their number (n = 34 soma were counted) is close to the number of VIH cells (n = 33), with a similar ratio between l-CHH cells (n = 21) and d-CHH cells (n = 13) of approximately 1.6 versus 1.3 for VIH cells. This figure fits well with the ratio of CHH ⁄ d-Phe3 CHH quantified in the lobster sinus glands [45; present study], which is not the case of VIH isomers, as noted earlier. On confocal mountings, subtle variations in colouration between d-CHH peri- karya were observed, although this much less pro- nounced than for VIH cells. This agrees with the results obtained in the crayfish Orconectes limosus, showing that l-isomer of CHH is always present in the different parts of the d-CHH cells, in decreasing amounts from the cell body to the axon terminal in the sinus gland, as a result of late and progressive isomerization of the Phe3 of the CHH during the migration of the secretion vesicles along the axonal tract [39,41]. The immunohistochemical and immunocytochemical results obtained in the present study invalidate our starting hypothesis proposing that the cells coexpress- ing VIH and CHH in the lobster X organ, as observed in previous studies [26,42], were actually producing the d-isomers of both hormones. Indeed, the lobster neu- roendocrine system is more complex than expected because, in addition to cells containing exclusively l-isomers of CHH or VIH, three types of cells were found, producing: (a) d-Phe3 CHH (d-CHH cells), (b) d-Trp4 VIH (d-VIH cells) or (c) a mixture of both C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4797 peptides (d-cells). Overall, five cell types producing CHH and VIH could be identified in the lobster X organ (Fig. 5). Nevertheless, it should be noted that d-CHH cells, d-VIH cells and d-cells may correspond to one single cell type producing a mixture of d-Trp4 VIH and d-Phe3 CHH in different proportions depending upon its physiological stage. Indeed, in situ hybridization in combination with immunohistochem- istry revealed that strong immunostaining of CHH and VIH may coincide with a weak or null mRNA label- ling and vice versa [26]. The existence of a sixth type of cell, producing a mixture of l-epimers of CHH and VIH had to been considered. It was researched by double labellings of successive ultrathin sinus glands sections (not shown), although this proved to be in vain. Similarly, no terminals exhibited simultaneous labelling for CHH and d-Trp4 VIH or VIH and d-Phe3 CHH (not shown). At present, it is not possible to assign a functional significance to the colocalization of the CHH and VIH epimers, especially because cellular colocalization of different neurohormones of the CHH family is not a general rule in the crustaceans. By contrast to the situ- ation in the lobster, VIH and CHH are synthesized and released by different cellular types in woodlouse [46] and in Norway lobster [47]. Similarly, moult- inhibiting hormone and CHH are localized in distinct neurosecretory pathways in several crabs [48], whereas colocalization of both peptides has been reported in prawns [49]. The presence, in the same cells and even in the same secretory granules, of peptides displaying a d-residue of different nature (Phe or Trp) and at different posi- tion (third or fourth) raises the question of the nature and characteristics of the enzyme(s) responsible for this modification. Indeed, only a few peptide isomerases, exhibiting very different substrate specificity, have been isolated so far, from the funnel-web spider A. aperta [50], the frog P. sauvagei [15] and from platypus venom [12,16]. Only structures of the first two are known, which appear to be totally unrelated because the frog enzyme presents similarities with the N-termi- nal H-domain of human IgG-Fc binding protein and the spider isomerase appears to belong to the serine protease family. Obviously, the large structural and functional divergences between these enzymes impede any speculation about the characteristics of the puta- tive lobster isomerase(s). Nevertheless, the occurrence, in lobster X organ cells, of two distinct enzymes with different substrate specificity and a variable expression pattern appears as a likely working hypothesis. Major challenges remaining for the future are the identification of the putative peptide isomerase(s) in crustaceans from these specialized cells from the X organ–sinus gland complex of the American lobster as well as the characterization of the receptors of CHH and VIH epimers, aiming to provide insights on the functional significance of the intriguing PTM that is l-tod-aminoacyl isomerization. Experimental procedures Animals and peptide purification H. americanus, weighing 300–500 g, were obtained from a commercial supplier (Metro, Bobigny, France). To reduce Fig. 5. General diagram of precursor pro- cessing of VIH and CHH isomers in relation to the different cell types in X organ–sinus gland complex. CPRP, CHH precursor- related peptide. a Amidation can be pre-, co- or post-cleavage of CPRP. b Cylization of CHH N-terminus is optional (N-terminus unblocked CHH can be released) and, simi- lar to isomerization, it occurs after CPRP cleavage. c By contrast to CHH, VIH is not N-terminal cyclized. L-CHH and L-VIH cells secrete exclusively CHH and VIH, respec- tively, whereas D-CHH and D-VIH cells release mainly the D-isomer of the respec- tive hormone, in addition to a variable amount of L-isomer. D cells secrete mainly the D-form of both CHH and VIH. Besides isomerization, the same PTMs occur in every type of CHH or VIH cell. Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al. 4798 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS their stress, lobsters were maintained in the laboratory for 2 weeks before experimentation, in filtered and re-circulat- ing artificial seawater at 13 °C. In the aquarium, hollow bricks provided shelters for the animals, which were fed weekly with mussels. The physiological status of the donors (sex, moulting and reproductive stage) was not recorded. The animals were anaesthetized on crushed ice for 30 min before dissection. VIH I and II were extracted from 30 H. americanus sinus glands (sinus gland equivalents, i.e.  1.5 lg or 160 pmol for VIH I and  2.7 lg or 280 pmol for VIH II). Peptides were extracted and purified as described previously [10]. HPLC fractions from the elution zone of CHHs and VIHs were collected manually to achieve optimal resolution between CHH and VIH isomers, respectively. HPLC frac- tions containing VIH I and II were dried under vacuum (Speed-Vac, Savant Instruments Inc., Holbrook, NY, USA). Enzymatic cleavage and digest fractionation Purified and dried VIHs (30 sinus gland equivalents) were redissolved in 5 lL of acetonitrile ⁄ water (1 : 1, v ⁄ v) and mixed with 0.4 lg of enzyme (Endoproteinase Asp-N sequencing grade, EC 3.4.24.33; Roche Diagnostics, Baˆ le, Switzerland) in 50 lL of phosphate buffer (50 mm, pH 8). After 22 h at 37 °C under stirring, the reaction was stopped by adding 5 lL of acetic acid (2 m). Then, the digests of each VIH were fractionated on a Nucleosil C18 (5 lm, 250 · 2.0 mm; Macherey-Nagel, Du ¨ ren, Germany) con- nected to the pump system and spectrophotometer. Peptides were eluted from the column by a gradient of acetonitrile in water at a flow-rate of 0.2 mLÆmin )1 . Both solvents con- tained trifluoroacetic acid (0.1% in water and 0.08% in ace- tonitrile). HPLC fractions from the elution zone of synthetic peptides (see below) were collected manually and dried under vacuum. Solid-phase peptide synthesis and antisera production Heptapeptides (Hep-l, Hep-dS2, Hep-dA3, Hep-dW4 and Hep-dF5) with the sequence corresponding to the N-termi- nus of lobster VIH and with all l -residues or a d-residue at different positions (Table 2) were synthesized using solid- phase FastMoc chemistry with a 433A Automated Peptide Synthesizer (Applied Biosystems, Foster City, CA, USA) as described previously [51]. The homogeneity of the synthetic peptides preparation was assessed by MALDI-TOF MS and analytical RP-HPLC. Decapeptides named Dec-l and Dec-dW4 (Table 2), cor- responding to the N-terminus of VIH I and II, respectively, were commercially synthesized (Eurogentec, Angers, France) and injected, after coupling to keyhole limpet haemocyanin, into rats (for Dec-l) or guinea pigs (for Dec-dW4) to generate polyclonal antisera. When necessary, specific IgG was purified according to a batch procedure that has been described previously [41]. MS Positive-ion mass spectra were recorded in reflectron mode with a single stage reflectron matrix-assisted laser desorp- tion ⁄ ionization time-of-flight (MALDI-TOF) mass spec- trometer (Voyager DE RP; Perseptive Biosystems Inc., Framingham, MA, USA) as described previously [51]. ELISA Specificity assays Direct ELISA was performed to determine the specificity of antisera. The wells of a plastic microtitre plate (Nunc, Ros- kilde, Denmark) were coated in triplicate with 100 ng of synthetic hepta- or decapeptides. In addition to the antisera r-anti-l (made in rat) and gp-anti-dW4 (produced in guinea pig), two antisera discriminating CHH isomers (gp-anti-pQl made in guinea pig and rb-anti- pQd in rabbit) [38] and an antiserum raised against purified VIH II (i.e. anti-4 pro- duced in guinea pig and recognizing both VIHs) [45] were used as primary antisera (Table 3). Secondary antibodies (anti-rat IgG, anti-guinea pig IgG and anti-rabbit IgG; all raised in goat and conjugated to alkaline-phosphatase; Sigma, Saint Louis, MO, USA) were used at 1 : 2000 dilu- tion (Table 3). Cross-reactivity of antisera between l- and d-peptides was calculated as the ratio between absorbance values obtained with the cross-reacting and the immunogen peptides. Analysis of native VIH Direct ELISA on RP-HPLC fractions from the elution zone of CHHs and VIHs from lobster sinus glands was per- formed: 10 lL aliquots of each fraction were pipetted in triplicate into the wells of a microtitre plate and dried under vacuum. The immunoassay was performed as Table 2. N-terminal amino acid sequence of vitellogenesis inhibit- ing hormone (VIH) and the synthetic peptides used in the present study. D-residues are indicated by bold and underlined letters. Peptide Sequence VIH ASAWFTNDECPG. Dec- L ASAWFTNDEC Dec- DW4 ASAWFTNDEC Hep- L ASAWFTN Hep- DS2 ASAWFTN Hep- DA3 ASAWFTN Hep- DW4 ASAWFTN Hep- DF5 ASAWFTN C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4799 [...]... (vitellogenesis inhibiting hormone) and CHH (crustacean hyperglycemic hormone) of the crustacean have the same precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster Homarus americanus Invert Reprod Dev 16, 43–52 43 Laverdure AM, Breuzet M, Soyez D & Becker J (1992) Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in. .. 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Characterization of crustacean hyperglycemic hormone from the crayfish (Procambarus clarkii): multiplicity of molecular forms by stereoinversion and diverse functions Gen Comp Endocrinol 95, 387–398 19 Serrano L, Charmantier G, Soyez D, Grousset E & Spanings-Pierrot C (2003) Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish... hyperglycemic and molt-inhibiting activity from sinus glands of the penaeid shrimp Penaeus vannamei Gen Comp Endocrinol 103, 41–53 37 Marco H, Brandt W & Gade G (1998) Elucidation of ¨ the amino acid sequence of a crustacean hyperglycemic hormone from the spiny lobster, Jasus lalandii Biochem Biophys Res Commun 248, 578–583 38 Soyez D, Laverdure AM, Kallen J & Van Herp F (1998) Demonstration of a cell-specific isomerization. .. crustacean hyperglycemic hormone J Biol Chem 269, 18295–18298 Soyez D, Lecaer JP, Noel PY & Rossier J (1991) Primary structure of two isoforms of the vitellogenesis inhibiting hormone from the lobster Homarus americanus Neuropeptides 20, 25–32 De Kleijn DPV, Sleutels FJGT, Martens GJM & Van Herp F (1994) Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus... 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Endocrinol 131, 134–142 Giulianini PG, Smullen R, Bentley MG & Ferrero EA (1998) Cytological and immunocytochemical study of the sinus gland in the Norway lobster Nephrops norvegicus Invert Reprod Dev 33, 57–68 Dircksen H & Soyez D (1998) The lobster thoracic ganglia-pericardial organ neurosecretory system: a large source of novel crustacean hyperglycemic hormone- like peptides In Proceedings of the1 9th... 47 48 49 50 51 52 53 Homarus americanus by in situ hybridization Gen Comp Endocrinol 87, 443–450 De Kleijn DPV, Janssen KPC, Waddy SL, Hegeman R, Lai WY, Martens GJM & VanHerp F (1998) Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus J Endocrinol 156, 291–298 Meusy JJ & Soyez D (1991) . Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family Ce ´ line Ollivaux 1,2,3 ,. decreasing amounts from the cell body to the axon terminal in the sinus gland, as a result of late and progressive isomerization of the Phe3 of the CHH during

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