Crustacean Hyperglycemic Hormone Precursor-related Peptide Research Articles

A novel crustacean hyperglycemic hormone (CHH) from the mud crab Scylla paramamosain regulating carbohydrate metabolism

Crustacean hyperglycemic hormone (CHH) plays a crucial role in regulating carbohydrate metabolism in crustaceans. In this study, a new cDNA encoding type I CHH peptide, termed Sp-CHH3, was isolated from the mud crab Scylla paramamosain and its potential functions were investigated. The full length cDNA of Sp-CHH3 was identified as encoding a 127-aa precursor composed of a 27-aa signal peptide, a 23-aa CHH precursor-related peptide and a 75-aa mature peptide with a typical motif of CHH. Phylogenic analysis suggested that, Sp-CHH3 is a previously unreported CHH from S. paramamosain. Tissue distribution analysis showed that Sp-CHH3 was mainly expressed in the eyestalk ganglia, thoracic ganglia, stomach and the ovary. A RNA interference experiments showed that after injection of Sp-CHH3-targeted dsRNA, both the level of Sp-CHH3 expression in the eyestalk ganglia and hemolymph glucose level decreased significantly. A further short-term starvation experiments demonstrated that, the level of Sp-CHH3 detected in the eyestalk ganglia was significantly up-regulated at the 12th h of starvation, it then fell back at the 24th h of starvation and subsequently remained relative stability between the 24th to 96th h of starvation. The hemolymph glucose level decreased significantly (P < .05) at each sampling time during the 96 h starvation duration when compared to that of 0 h (prior to starvation) and the overall trend was largely correlated with the level of Sp-CHH3 expression in the eyestalk ganglia. In summary, the results suggest that Sp-CHH3 plays a functional role in regulating carbohydrate metabolism in S. paramamosain.

Prediction of a neuropeptidome for the eyestalk ganglia of the lobster Homarus americanus using a tissue-specific de novo assembled transcriptome

In silico transcriptome mining is a powerful tool for crustacean peptidome prediction. Using homology-based BLAST searches and a simple bioinformatics workflow, large peptidomes have recently been predicted for a variety of crustaceans, including the lobster, Homarus americanus. Interestingly, no in silico studies have been conducted on the eyestalk ganglia (lamina ganglionaris, medulla externa, medulla interna and medulla terminalis) of the lobster, although the eyestalk is the location of a major neuroendocrine complex, i.e., the X-organ-sinus gland system. Here, an H. americanus eyestalk ganglia-specific transcriptome was produced using the de novo assembler Trinity. This transcriptome was generated from 130,973,220 Illumina reads and consists of 147,542 unique contigs. Eighty-nine neuropeptide-encoding transcripts were identified from this dataset, allowing for the deduction of 62 distinct pre/preprohormones. Two hundred sixty-two neuropeptides were predicted from this set of precursors; the peptides include members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon α, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, glycoprotein hormone β5, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families. The predicted peptides expand the H. americanus eyestalk ganglia neuropeptidome approximately 7-fold, and include 78 peptides new to the lobster. The transcriptome and predicted neuropeptidome described here provide new resources for investigating peptidergic signaling within/from the lobster eyestalk ganglia.

Neuropeptidergic Signaling in the American Lobster Homarus americanus: New Insights from High-Throughput Nucleotide Sequencing.

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without the assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems. Mass spectrometry (MS)-based neuropeptidomics aims to completely characterize the neuropeptides in a target organism as an important first step toward a better understanding of the structure and function of these complex signaling molecules. Although liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition is a powerful tool in peptidomic research, it often lacks the capability for de novo sequencing of mid-size and large peptides due to inefficient fragmentation of peptides larger than 4kDa. This study describes a multi-scale strategy for complete and confident sequence elucidation of various sizes of neuropeptides in the crustacean nervous system. The aim is to fill a technical gap where the conventional strategy is inefficient for comprehensive characterization of a complex neuropeptidome without assistance of genomic information. Nine novel neuropeptides in a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ of the spiny lobster, P. interruptus. The resulting molecular information extracted from such multi-scale peptidomic analysis will greatly accelerate functional studies of these novel neuropeptides.

Crustacean hyperglycemic hormone precursor transcripts in the hemocytes of the crayfish Procambarus clarkii: Novel sequence characteristics relating to gene splicing pattern and transcript stability

It was demonstrated in a previous study (Wu et al., 2012b) that crustacean hyperglycemic hormone (CHH) gene is expressed in the hemocyte of Procambarus clarkii. In the present study, 2 additional cDNAs (CHH2-L and tCHH2) from the hemocyte and a CHH gene (CHH2) from the abdominal muscle of the same species were cloned. Analyses of the cDNA and genomic sequences suggested that, similar to other previously reported CHH genes, 2 precursor transcripts (CHH2 and CHH2-L) would be derived from CHH2 gene through a process of RNA alternative splicing, and CHH2 and CHH2-L each encode a precursor containing a signal peptide, a CHH precursor-related peptide, and a mature peptide. Further, tCHH2 sequence consists of exon I, exon II, and a truncated segment of intron II of CHH2 gene, followed by a previously unknown 3′sequence. It is suggested that, because the truncation disrupts the highly conserved RNA splice acceptor site, the truncated segment is retained within tCHH2, resulting in encoding a precursor containing the typical precursor components except the mature peptide is truncated with only 40 residues. In addition, unlike 2 other previously identified transcripts (referred to as CHH1 and CHH1-L), CHH2-L, CHH2, tCHH2 contain in the 3′-UTRs 3–5 AU-rich elements (AREs). The data showed that multiple CHH genes are expressed in crayfish hemocytes. Novel sequence characteristics of the transcripts result in an RNA splicing pattern that yields a transcript (tCHH2) encoding a precursor with an atypical truncated mature peptide and possibly leads to a different expression dynamics of the precursors encoded by the ARE-containing transcripts.

Cloning and molecular structure analysis of crustacean hyperglycemic hormone(Ers-CHH)in Eriocheir sinensis

Crustacean hyperglycemic hormone(CHH)is a peptide hormone originally identified in a crustacean neurosecretory complex,the X-organ/sinus gland complex,and located within the eyestalks.CHH is placed in the crustacean hyperglycemic hormone family,which also includes molt-inhibiting hormone(MIH),vitellogenesis-inhibiting hormone(VIH)or gonad-inhibiting hormone(GIH),mandibular organ-inhibiting hormone(MOIH)and insect ion transport peptide(ITP).CHH plays a significant role in the growth,molting and reproduction of crustaceans.By using degenerate primers which were designed according to the N-terminal amino acid sequences of CHH isolated by our lab and the sequences of homologous species CHH,a novel cDNA of crustacean hyperglycemic hormone gene(Ers-CHH,GenBank Accession No:JX485664)was cloned from Chinese mitten crab(Eriocheir sinensis).The full length cDNA consists of 585 bp with a 453 bp open reading frame,encoding 150 amino acids,containing a 30 amino acids signal peptide,and a 42 amino acids CHH precursor-related peptide,a 78 amino acids mature peptide.The mature peptide contains 6 conserved cysteines which formed three disulfide bonds C7-C43、C23-C39、C26-C52.There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.The three-dimensional structure prediction showed that Ers-CHH mature peptide consisted of 4 α-helixes,and not with the β-sheet structure.5 Prosite patterns,which were analyzed by a Swiss-PdbViewer(v4.0.4),are P5:Protein kinase C phosphorylation site(Ser6,Lys8);P6:Casein kinase Ⅱ phosphorylation site(Ser17,Glu20);P8:N-myristoylation site(Gly38,Cys39,Asn42,Cys43);PS00342:Microbodies C-terminal targeting signal(Ser17,Lys18,Leu19);PS01250:Arthropod CHH/MIH/GIH neurohormones family(Val35,Cys39,Arg40,Asn42,Cys43,Tyr44,Asn46,Phe49,Cys52).The results of the structural model comparison show that the structure of Ers-CHH has high similarity with other CHHs and Ers-CHH.They have no β-sheet structure,except that CHH has 4α-helixes,MIH has 5α-helixes.Although α1 helix of MIH could not be found in CHHs,the position and the amino acid number of α2-α5 helixes in MIH molecular were same as the α1-α4 helixes in CHH.It might be able to interpret the function similarity between CHH and MIH.Multiple alignment results showed that Ers-CHH has the highest identity with Ptychognathus pusillus CHH(92%),and it also shared high identities with Neohelice granulata(86%)and Pachygrapsus marmoratus(72%).However,it showed lower identity with CHH from crayfishes,such as Nephrops norvegicus(49%)and Homarus gammarus(46%).Multiple alignments of Ers-CHH and MIH from other crustaceans showed that only six amino acid residues(Arg13,Asp25,Asn28,Arg31,Arg40,Phe49)in Ers-CHH sequence were same with MIH sequences.It may be helpful for further research on regulation mechanism of CHH in the process of crab growth and carbohydrate metabolism.

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): Cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.

Molecular cloning of a putative crustacean hyperglycemic hormone (CHH) isoform from extra-eyestalk tissue of the blue crab ( Callinectes sapidus), and determination of temporal and spatial patterns of CHH gene expression

Crustacean hyperglycemic hormone (CHH) is a polypeptide neurohormone involved in regulation of multiple physiological processes. We report here the cloning from thoracic ganglia of the blue crab ( Callinectes sapidus) a cDNA ( CsCHH-2) encoding a putative CHH isoform (CsCHH-2). CsCHH-2 is structurally similar to a putative preproCHH (CsCHH-1) previously cloned from eyestalk ganglia of C. sapidus. The two preprohormones possess an identical signal peptide and CHH precursor related peptide, but differ in the mature CHH polypeptide. An analysis by RT-PCR of the tissue distribution of CsCHH-1 and CsCHH-2 revealed the former is restricted to eyestalk neural ganglia, while the latter is widely distributed among tissues. The type of CHH transcript present in eyestalk and thoracic ganglia did not vary as a function of the molt cycle. An assessment of transcript abundance in tissues of intermolt crabs showed the abundance of the CsCHH-1 transcript in eyestalk ganglia far exceeds the abundance of the CsCHH-2 transcript in extra-eyestalk tissue. An assessment of transcript abundance during a molt cycle showed CsCHH-1 transcript abundance in eyestalk ganglia was low during intermolt, rose during premolt, reaching a peak in D 3, then fell prior to molting, and remained low during postmolt. By contrast, CsCHH-2 transcript abundance in thoracic ganglia was low during intermolt, rose sharply during D 2, then dropped in D 3 and remained low during postmolt. The results are consistent with the hypothesis that CsCHH-1 and CsCHH-2 differ with respect to physiological function.

Expanding the Crustacean Neuropeptidome Using a Multifaceted Mass Spectrometric Approach

Jonah crab Cancer borealis is an excellent, long-served model organism for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), MALDI time-of-flight (TOF)/TOF MS and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS). To enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including FMRFamide-related peptide (FaRP), allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.

Molecular cloning and differential expression pattern of two structural variants of the crustacean hyperglycemic hormone family from the mud crab Scylla olivacea

Two full-length cDNA sequences encoding a crustacean hyperglycemic hormone (CHH) precursor were cloned from tissues of the mud crab Scylla olivacea. Sco-CHH ( S. olivacea CHH) was cloned from eyestalk ganglia, whereas Sco-CHH-L ( S. olivacea CHH-like peptide) was cloned from extra-eyestalk tissues (pericardial organ and thoracic ganglia). Each conceptually translated precursor is expected to be processed into a signal peptide, a CHH precursor-related peptide (CPRP), and a mature CHH or CHH-like peptide. The two precursors are identical in amino acid sequence through the 40th residue of the mature peptide, but different from each other substantially in the C-terminus. Both CHH variants contain the six highly conserved cysteine residues characteristic of the CHH family peptides, and share higher sequence identities with other brachyuran CHH sequences than with those of other taxonomic groups. As determined by reverse transcription-polymerase chain reaction (RT-PCR), the transcripts of Sco-CHH and Sco-CHH-L were present in eyestalk ganglia and several extra-eyestalk tissues (the thoracic ganglia, pericardial organ, brain, circumesophageal connectives, and gut). Sco-CHH was the predominant form in eyestalk ganglia, while Sco-CHH-L was the predominant form in several extra-eyestalk tissues. Neither transcript was expressed in the muscle, hepatopancreas, ovary, testis, heart, or gill. Antisera were raised against synthetic peptides corresponding to a stretch of sequence-specific to the C-terminus of Sco-CHH or Sco-CHH-L. Western blot analyses of tissues expressing Sco-CHH and Sco-CHH-L detected a Sco-CHH immunoreactive protein in the sinus gland, and a Sco-CHH-L immunoreactive protein in the pericardial organ. Immunohistochemical analyses of the eyestalk ganglia localized both Sco-CHH and Sco-CHH-L immunoreactivity to the sinus gland, and only Sco-CHH immunoreactivity to the X-organ somata; analyses of the pericardial organs also localized both Sco-CHH and Sco-CHH-L immunoreactivity to the anterior and posterior bars, as well as to longitudinal trunks joining the two bars. The combined data provided supporting evidence that Sco-CHH and Sco-CHH-L are co-localized in the same tissue.

Molecular cloning of four cDNAs encoding prepro-crustacean hyperglycemic hormone (CHH) from the eyestalk of the red rock crab Cancer productus: Identification of two genetically encoded CHH isoforms and two putative post-translationally derived CHH variants

Recently, we demonstrated that the four known sinus gland (SG) isoforms of Cancer productus crustacean hyperglycemic hormone precursor-related peptide (Capr-CPRP I–IV) are differentially distributed in conserved patterns among individual crabs. This finding strongly supported the presence of multiple prepro-crustacean hyperglycemic hormone ( chh) transcripts in each crab, as well as the translation and processing of the encoded prepro-hormones. Whether these transcripts contained common or distinct isoforms of CHH remained unknown. To address this question, molecular analyses of the C. productus eyestalk prepro-chhs were undertaken. Using a PCR-based cloning strategy, four prepro-chh cDNAs were characterized: one encoding CPRP I, one encoding CPRP III (found to possess Ile 26 rather than Leu 26 as reported previously), and two encoding CPRP II. No cDNA encoding CPRP IV was identified. The deduced CHH present in the prepro-hormones containing CPRP I and III were identical (Capr-CHH I) and differed from that (Capr-CHH II) present in the two prepro-hormones containing Capr-CPRP II at a single residue, a Thr 5 for Ser 5 substitution. As both CHH isoforms possess Glu at position 1, a cyclization of this residue to pyroglutamine is likely as the peptides mature, as has been seen for the CHHs of other brachyuran species. Likewise, homology to other CHHs suggests all C. productus isoforms are C-terminally amidated. These post-translational modifications would result in four SG isoforms of CHH: Capr-CHH I, Capr-pyro-CHH I, Capr-CHH II, and Capr-pyro-CHH II. Southern blotting supported the hypothesis that at least three prepro-chh transcripts are present in each crab, while dual in situ hybridization-immunohistochemistry localized the transcripts to previously mapped CHH immunopositive somata in the X-organ, the major source of innervation to the SG.

Direct tissue MALDI-FTMS profiling of individual Cancer productus sinus glands reveals that one of three distinct combinations of crustacean hyperglycemic hormone precursor-related peptide (CPRP) isoforms are present in individual crabs

Over the past decade, mass spectrometry has become a prominent technique for identifying peptide hormones. In crustaceans, studies directed at characterizing the peptide complements present in neuroendocrine structures have generally involved the isolation of tissue from a large number of individuals, which are pooled, extracted, purified, and then analyzed via chromatographic techniques coupled with mass spectrometry. While this approach provides information on the peptides present in the population of animals used as the tissue source, data on the peptide complement present in any individual animal are lost. Direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) of single tissues has the potential to identify differences in peptide expression between individuals. Here, we have used direct tissue MALDI-FTMS of individual sinus glands (SGs) to show that the four isoforms of crustacean hyperglycemic hormone precursor-related peptide (CPRP) identified previously from pooled Cancer productus SGs ( i.e. Fu, Q., Christie, A.E., Li, L. 2005. Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus. Peptides 26, 2137–2150.) are differentially distributed in conserved patterns among individual crabs. Of the crabs examined, ∼61% of the individuals possessed Capr-CPRP I and II, but not III or IV, ∼26% Capr-CPRP I, II and III, but not IV, and ∼13% Capr-CPRP I, II and IV, but not III. Our findings set the stage for future molecular investigations on the origin(s) of this individual-specific variation in CPRP complement, as well as investigations of the function and regulation of the individual isoforms. These data also lend a cautionary note to the assumption that the peptides identified via pooled tissues reveal an accurate picture of the peptides present in any given individual.

Members of the crustacean hyperglycemic hormone (CHH) peptide family are differentially distributed both between and within the neuroendocrine organs ofCancercrabs: implications for differential release and pleiotropic function

The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH, moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancer suggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Molecular cloning of a cDNA encoding a crustacean hyperglycemic hormone from eyestalk ganglia of the blue crab, Callinectes sapidus

Crustacean hyperglycemic hormone (CHH), a polypeptide with multiple physiological effects, was first identified in the X-organ/sinus gland neurosecretory system of the eyestalks. In studies reported here, we used a PCR-based cloning strategy (RT-PCR followed by 5′- and 3′-RACE) to clone from blue crab ( Callinectes sapidus) eyestalk ganglia a cDNA ( CsCHH-1) encoding a putative CHH preprohormone. Sequence analysis revealed the preprohormone included all structural features previously reported for CHH preprohormones: a signal peptide, a CHH precursor-related peptide (CPRP), the CHH polypeptide, and a C-terminal basic processing site. Further, the deduced amino acid sequence of the mature polypeptide included all signature domains previously reported for CHH. The primary structure of blue crab CHH is most closely related to CHH from other brachyurans. RT-PCR revealed the CsCHH-1 transcript was present in eyestalk ganglia, but was undetectable in other tissues tested. A transcript encoding a similar CHH-like preprohormone was detected in thoracic ganglion, ventral nerve cord, and brain, but was not detected in eyestalk ganglia.

Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas: A possible osmoregulatory role

To determine the possible involvement of crustacean hyperglycemic hormone (CHH) in osmoregulation in crustaceans, ligand binding and second messenger assays were performed on gills and hindgut preparations of the green shore crab Carcinus maenas, whilst midgut gland, previously known as one of the target tissues of CHH served as a control tissue. Classical receptor binding analyses using [ 125I]CHH by saturation and displacement experiments from membrane preparations from gills, hindgut, and midgut glands demonstrated that CHH binding characteristics involved one site, highly specific, saturable, and displaceable kinetics: (gills: K D 5.87 ± 2.05 × 10 −10 and B MAX 6.50 ± 1.15 × 10 −10, hindgut: K D 3.54 ± 1.49 × 10 −10 and B MAX 2.31 ± 0.44 × 10 −10, and midgut gland: K D 7.28 ± 0.9 × 10 −10 and B MAX 3.28 ± 0.25 × 10 −10) all expressed as M/mg protein. No differences, in terms of displacement were observed between the two CHH isoforms (N-terminally blocked pGlu and unblocked Gln) variants. CHH binding sites appeared to be coupled to a second messenger system involving cGMP in all the tissues examined. Exposure of crabs to dilute seawater increased levels of cGMP, glucose in gills and circulating CHH levels. Other crustacean neuropeptides including crustacean cardioactive peptide, molt inhibiting hormone, L-enkephalin, FMRF-amide, proctolin, and crustacean hyperglycemic hormone precursor-related peptide were tested with regard to possible osmoregulatory roles with reference to changes in second messenger (cAMP and cGMP) concentrations in gill, hindgut, and midgut tissues in vitro, following application at 2 × 10 −8 M but all were found to be inactive. Thus, it seems likely that CHH is a pertinent neurohormone involved in osmoregulation, thus expanding its many functions as a pleiotropic hormone in crustaceans.

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 1 1 The sequences of PamCHHA XO, PamCHHB XO and PamCHHB PO have been included in the GenBank sequence database with accession numbers AY180333, AY180334 and AY180335 respectively. and the fresh water crab Potamon ibericum. 2 2 The sequences of PoiCHH XO and PoiCHH PO have been included in the GenBank sequence database with accession numbers DQ176431 and DQ176432 respectively. 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.

Identification of Neuropeptides from the Sinus Gland of the Crayfish Orconectes limosus Using Nanoscale On-line Liquid Chromatography Tandem Mass Spectrometry

In this article, a novel and sensitive analytical strategy for direct characterization of neuropeptides from the X-Organ-sinus gland neurosecretory system of the crayfish Orconectes limosus is presented. A desalted extract corresponding to 0.5 sinus gland equivalents was analyzed in a nanoflow liquid chromatography system coupled to quadrupole time-of-flight tandem mass spectrometry (nanoLC-QTOF MS/MS). The existence and structural identity of four crustacean hyperglycemic hormone precursor-related peptide variants and two new genetic variants of the pigment-dispersing hormone, not detected by conventional chromatographic systems, molecular cloning, or immunochemical methods before, was revealed. The here-presented approach of the combined LC-QTOF MS/MS technique is a powerful tool to discover new peptide hormones in biological systems, due to its sensitivity, accuracy, and speed.

Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives

I present an overview of recent research on the isolation and characterization of members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. Members of this arthropod-specific family include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). There are two subfamilies of this neuropeptide group, based upon the presence or absence of a C-terminal CHH precursor-related peptide. There are also sequence motif differences between these subfamilies. Most of the peptides comprising this neuropeptide family are synthesized and released by the eyestalk X-organ/sinus gland complex. Recent experiments have demonstrated the presence of extra-eyestalk cells that produce CHH and the assignment of additional functions to this hormone family.

Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives1

I present an overview of recent research on the isolation and characterization of members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. Members of this arthropod-specific family include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). There are two subfamilies of this neuropeptide group, based upon the presence or absence of a C-terminal CHH precursor-related peptide. There are also sequence motif differences between these subfamilies. Most of the peptides comprising this neuropeptide family are synthesized and released by the eyestalk X-organ/sinus gland complex. Recent experiments have demonstrated the presence of extra-eyestalk cells that produce CHH and the assignment of additional functions to this hormone family.

Five Crustacean Hyperglycemic Family Hormones of Penaeus monodon: Complementary DNA Sequence and Identification in Single Sinus Glands by Electrospray Ionization-Fourier Transform Mass Spectrometry.

Five novel neuropeptides, designated Pm-sgp-I to -V, of the crustacean hyperglycemic hormone (CHH) family have been identified from the giant tiger prawn Penaeus monodon by isolation of the preprohormone genes from an eyestalk complementary DNA library. On the basis of sequence similarity, the encoded peptides have been classified as CHH-like type I hormones, which include all known CHHs and the molt-inhibiting hormone (MIH) of the lobster Homarus americanus. Consistent with CHH type I preprohormones, the Pm-sgp precursors include a signal peptide, a CHH precursor-related peptide (CPRP), and the CHH-like hormone. Analysis by electrospray ionization-Fourier transform mass spectrometry enabled the neuropeptide complement of individual sinus glands to be resolved. It also confirmed the presence of the five Pm-sgp neuropeptides within the sinus gland of an individual animal, in that the masses observed were consistent with those predicted from the gene sequence of the Pm-sgps after posttranslational modification. These modifications included cleavage of the signal peptide and precursor protein, carboxy-terminal amidation, and formation of three disulfide bridges. Analysis of crude extracts of single sinus glands from different animals revealed variation in neuropeptide content and will provide a tool for determining whether the content varies as a function of the physiological state of the animal.