Biogenesis of D-amino acid containing peptides/proteins: where, when and how?

Peptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation.

Natural and synthetic chiral isoforms of crustacean hyperglycemic hormone from the crayfish Astacus leptodactylus: hyperglycemic activity and hemolymphatic clearance.

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose-response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues.

Experimental strategies for the analysis of D-amino acid containing peptides in crustaceans: a review.

Detection of D-amino acids in natural peptides has been, and remains a challenging task, as peptidyl isomerization is a peculiar and subtle posttranslational modification that does not induce any change in primary sequence or in physicochemical properties of the molecule such as molecular mass or pI. Therefore, the presence of a D-amino acid residue in a peptide chain is generally transparent to classical methods of peptide analysis (electrophoresis, chromatography, mass spectrometry, molecular biology). In this article, we will review the various experimental strategies and analytical techniques, which have been used to characterize and to study D-amino acid containing peptides in crustaceans.

Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans.

BACKGROUND: Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated. RESULTS: CHH family peptides were characterized by molecular cloning in a branchiopod crustacean, Daphnia magna, and in a collembolan, Folsomia candida. Genes encoding such peptides were also rebuilt in silico from genomic sequences of another branchiopod, a chelicerate and two nematodes. These sequences were included in updated datasets to build phylogenies of the CHH family in pancrustaceans. These phylogenies suggest that peptides found in Branchiopoda and Collembola are more closely related to insect ITPs than to crustacean CHHs. Datasets were also used to support a phylogenetic hypothesis about pancrustacean relationships, which, in addition to gene structures, allowed us to propose two evolutionary scenarios of this multigenic family in ecdysozoans. CONCLUSIONS: Evolutionary scenarios suggest that CHH family genes of ecdysozoans originate from an ancestral two-exon gene, and genes of arthropods from a three-exon one. In malacostracans, the evolution of the CHH family has involved several duplication, insertion or deletion events, leading to neuropeptides with a wide variety of functions, as observed in decapods. This family could thus constitute a promising model to investigate the links between gene duplications and functional divergence.

Characterization of a membrane-bound angiotensin-converting enzyme isoform in crayfish testis and evidence for its release into the seminal fluid.

In the present study, an isoform of angiotensin-converting enzyme was characterized from the testis of a decapod crustacean, the crayfish Astacus leptodactylus. Angiotensin-converting enzyme cDNA, obtained by 3'- to 5' RACE of testis RNAs, codes for a predicted one-domain protein similar to the mammalian germinal isoform of angiotensin-converting enzyme. All amino acid residues involved in enzyme activity are highly conserved, and a potential C-terminus transmembrane anchor may be predicted from the sequence. Comparison of this testicular isoform with angiotensin-converting enzyme from other crustaceans, namely Carcinus maenas, Homarus americanus (both reconstituted for this study from expressed-sequence tag data) and Daphnia pulex, suggests that membrane-bound angiotensin-converting enzyme occurs widely in crustaceans, conversely to other invertebrate groups where angiotensin-converting enzyme is predominantly a soluble protein. In situ hybridization and immunohistochemistry performed on testis sections show that angiotensin-converting enzyme mRNA is mainly localized in spermatogonias, whereas protein is present in spermatozoids. By contrast, in vas deferens, immunoreactivity is detected in the seminal fluid rather than in germ cells. Accordingly, angiotensin-converting enzyme activity assays of testis and vas deferens extracts demonstrate that the enzyme is present in the membrane fraction in testis, but in the soluble fraction in vas deferens. Taken together, the results obtained in the present study suggest that, during the migration of spermatozoids from testis to vas deferens, the enzyme is cleaved from the membrane of the germ cells and released into the seminal fluid. To our knowledge, this present study is the first to report such a maturation process for angiotensin-converting enzyme outside of mammals.

Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family.

D-aminoacyl residues have been detected in various animal peptides from several taxa, especially vertebrates and arthropods. This unusual polymorphism 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- or a D- 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.

D-amino acid detection in peptides by MALDI-TOF-TOF.

Detection of a D-amino acid residue in natural peptides by mass spectrometry remains a challenging task, as this post-translational modification does not induce any change in molecular mass. To our knowledge, the present article is the first report using matrix-assisted laser desorption/ionization (MALDI) for the discrimination and the quantification of peptide isomers. In this work, we used synthetic hepta- and decapeptides of biological relevance and their isomers. All-L sequences and some isomers containing a D-residue in various positions were analyzed.

New insights into evolution of crustacean hyperglycaemic hormone in decapods--first characterization in Anomura.

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.

Evidence for an angiotensin-converting enzyme (ACE) polymorphism in the crayfish Astacus leptodactylus.

The present study was initiated to characterize angiotensin-converting enzyme (ACE) in Crustaceans. Using degenerate DNA primers deduced from consensus sequences located upward and downward from the active site of ACEs from different arthropod species, several tissues from the crayfish Astacus leptodactylus were screened by RT-PCR. Amplicons were obtained from hepatopancreas, testis and hemocytes. Analysis of the predicted protein sequences after cloning and Northern blot experiments revealed an original and complex polymorphism of the ACE-like active site. Two variants were obtained in the hepatopancreas, one displaying a 6.4 kb size transcript, probably corresponding to a double domain ACE, with an unusual active site structure while the other had a transcript size of 2.5 kb, close to the size of the transcript obtained in testis and hemocytes (2 and 3kb, respectively), likely representing single domain enzymes. Functional assays using a synthetic substrate were performed from the different tissues and showed a maximal ACE-like activity associated to membrane fraction from testis and hepatopancreas.

Crustacean hyperglycemic and vitellogenesis-inhibiting hormones in the lobster Homarus gammarus.

Crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH), produced by the X organ-sinus gland neurosecretory complex, belong to a peptide group referred to as the CHH family, which is widely distributed in arthropods. In this study, genetic variants and post-translationally modified isoforms of CHH and VIH were characterized in the European lobster Homarus gammarus. With the use of RP-HPLC and ELISA with specific antibodies that discriminate between stereoisomers of CHH and VIH, two groups of CHH-immunoreactive peaks were characterized from HPLC fractions of sinus gland extract (CHH A and CHH B); each group contained two variants (CHH and D-Phe3CHH). In the same way, two VIH-immunoreactive peaks (VIH and D-Trp4VIH) were demonstrated in HPLC fractions from sinus gland extract. The masses of these different neuropeptides were determined by FT-ICR MS: CHH A and CHH B spectra exhibited monoisotopic ions at 8557.05 Da and 8527.04 Da, respectively, and both VIH isomers displayed an m/z value of 9129.19 Da. Two full-length cDNAs encoding preprohomones of CHH A and CHH B and only one cDNA for VIH precursor were cloned and sequenced from X organ RNA. Comparison of CHH sequences between European lobster and other Astacoidea suggests that the most hydrophobic form appeared first during crustacean evolution.

The crustacean hyperglycemic hormones from an euryhaline crab Pachygrapsus marmoratus and a fresh water crab Potamon ibericum: eyestalk and pericardial isoforms.

The structures of crustacean hyperglycemic hormones (CHH) were investigated in two crabs, the coastal euryhaline crab Pachygrapsus marmoratus and the fresh water crab Potamon ibericum. The neuropeptide mRNAs were extracted from pericardial and X-organs (PO and XO), and the sequences of the cDNA encoding the hormones' precursors were determined. The X-organ preprohormones are composed of 29 and 28 amino acid signal peptides in P. marmoratus and P. ibericum respectively, followed by 43 and 41 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP) flanking the 72 amino acid crustacean hyperglycemic hormones. A similar organization is reported for pericardial preprohormones with identical sequences for the signal peptide, the CPRP and the N-terminal sequences of CHH (1-40), but remaining sequences (41-72 and 41-71) differing considerably. In P. marmoratus two CHH cDNAs were characterized from XO and evidences were obtained for the existence of at least two forms in the PO. From our results and by comparison with other known sequences, a consensus pattern for crab pericardial CHH could be pointed out. Analysis of the data presented in this article using phylogenetic methods reveals that the two crab species studied are much closer than previously predicted.

Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry.

Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway.

Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.

This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na(+) concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)-related peptides, including stereoisomers (L-CHH and D-Phe(3) CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe(3) CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe(3) CHH fraction significantly increased the hemolymph osmolality and Na(+) content 24 h after injection. Two other CHH-related peptides caused a smaller increase in Na(+) concentration. No significant variation was observed in hemolymph Cl(-) concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe(3) CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.

Enkephalinergic control of the secretory activity of neurons producing stereoisomers of crustacean hyperglycemic hormone in the eyestalk of the crayfish Orconectes limosus.

A subgroup of neurons in the classical X organ sinus gland neuroendocrine system of the crayfish (Orconectes limosus) eyestalk produces two chiral forms of the crustacean hyperglycemic hormone (CHH) in two different types of neurons: CHH in 22 cells and D Phe(3) CHH in eight cells. Previous reports have demonstrated that release of CHH from the sinus gland is inhibited by enkephalins. Here, we have addressed the questions of 1) whether this inhibition affects one or both types of CHH neurons, 2) where the site of enkephalinergic control of CHH and/or D Phe(3) CHH is, and 3) whether the inhibitory effect is due to direct or indirect interactions of enkephalinergic neurons with CHH cells. In vitro incubations of neurosecretory complexes followed by immunoassays of CHH isoforms indicated that both methionine and leucine enkephalins inhibit release of the two CHH isoforms from crayfish eyestalks, by a receptor mediated process. Whole mount double or triple immunofluorescence labelings combined with confocal microscopy revealed enkephalin immunostaining in all neuropils of the eyestalk, except in the sinus gland. Virtual thin confocal sections showed many close appositions between terminals of enkephalinergic neurons and dendritic arborizations of specific CHH immunoreactive cells in the medulla terminalis neuropil. This provides the first evidence for direct inputs from enkephalinergic neurons into dendrites of both CHH cell types, which suggests that enkephalins inhibit release of both CHH isoforms via synaptic contacts.

Structure and phylogeny of the crustacean hyperglycemic hormone and its precursor from a hydrothermal vent crustacean: the crab Bythograea thermydron.

The structure of a well-known neurohormone involved in homeostasis regulation and stress response, the crustacean hyperglycemic hormone, was investigated in the deep-sea hydrothermal vent crab Bythograea thermydron. The neuropeptide was isolated from neurohemal organs (sinus glands) and its biological activity checked using an homologous bioassay. Partial amino acid sequence was established by a combination of Edman chemistry and mass spectrometry. Then, the sequence of the cDNA encoding the hormone precursor was determined. The preprohormone is composed of a 29 amino acid signal peptide, followed by a 41 amino acid associated peptide flanking the 72 amino acid hyperglycemic hormone. Comparison of these data with other known crab hyperglycemic hormone and prohormone sequences was performed using phylogenetic analysis methods.