Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives

I present an overview of recent research on the isolation andcharacterization of members of the crustacean hyperglycemichormone (CHH) neuropeptide family. Members of this arthropod-specificfamily include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibitinghormone (VIH), and mandibular organ-inhibiting hormone (MOIH).There are two subfamilies of this neuropeptide group, basedupon the presence or absence of a C-terminal CHH precursor-relatedpeptide. There are also sequence motif differences between thesesubfamilies. Most of the peptides comprising this neuropeptidefamily are synthesized and released by the eyestalk X-organ/sinusgland complex. Recent experiments have demonstrated the presenceof extra-eyestalk cells that produce CHH and the assignmentof 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. Received March 26, 1999; accepted September 10, 1999.     

cDNA cloning of a mandibular organ inhibiting hormone from the spider crabLibinia emarginata

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crabLibinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks ofLibinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.     

甲壳动物高血糖激素家族生理功能研究进展

甲壳动物高血糖激素家族是甲壳动物特有的神经多肽激素家族,主要由眼柄的X-器窦腺复合体(XO-SG)合成与分泌,包括高血糖激素(CHH)、蜕皮抑制激素(MIH)、性腺抑制激素(GIH)和大颚器抑制激素(MOIH),协同调控着甲壳动物的生长、繁殖与蜕皮等生理生化过程.本文就目前CHH家族神经肽的功能研究,包括功能研究的方法、各个激素的功能以及分泌调控等研究进展作一综述.     

The solution structure of molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus

Molting in crustaceans is controlled by molt-inhibiting hormone (MIH) and ecdysteroids. It is presumed that MIH inhibits the synthesis and the secretion of ecdysteroids by the Y-organ, resulting in molt suppression. The amino acid sequence of MIH is similar to that of crustacean hyperglycemic hormone (CHH), and therefore, they form a peptide family referred to as the CHH family. Most of the CHH family peptides show no cross-activity, whereas a few peptides show multiple hormonal activities. To reveal the structural basis of this functional specificity, we determined the solution structure of MIH from the Kuruma prawn Marsupenaeus japonicus and compared the solution structure of MIH with a homology-modeled structure of M. japonicus CHH. The solution structure of MIH consisted of five alpha-helices and no beta-structures, constituting a novel structural motif. The homology-modeled structure of M. japonicus CHH was very similar to the solution structure of MIH with the exception of the absence of the N-terminal alpha-helix and the C-terminal tail, which were sterically close to each other. The surface properties of MIH around this region were quite different from those of CHH. These results strongly suggest that this region is a functionally important site for conferring molt-inhibiting activity.     

Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

The crustacean hyperglycemic hormone (CHH) peptides regulate diverse physiological processes from reproduction to metabolism and molting in arthropods. In insects, the ion transport peptides (ITP), also members of the CHH family, have only been implicated in ion transport. In this study, we sequenced a nucleotide fragment spanning the conserved A1/A2 region of the putative CHH/ITP gene. This fragment was amplified from larval cDNA of the tobacco hornworm, Manduca sexta and showed a high degree of sequence conservation with the same region from other insects and, to a lesser degree, with that of crustacean species, suggesting the presence of a Manduca-specific CHH/ITP mRNA (MasITP mRNA). CHH-like immunocytochemical analyses with two crustacean antisera (from Carcinus maenas and Cancer pagurus) identified the presence of CHH-like immunoreactivity in nervous tissue of all developmental stages, but not in the gut of M. sexta. Specifically, CHH-like peptides localized to paired type IA₂ neurosecretory cells of the pars lateralis of the brain (projecting ipsilaterallly to the corpora cardiaca-allata complex) and to neurosecretory cells and transverse nerves of the ventral nerve cord in larvae, pupae, and adults. The distribution of the putative MasITP peptide shifted during development in a manner consistent with metamorphic reorganization. A comparison of hemolymph equivalents of CHH detected by enzyme-linked immunosorbent assay with CHH-like immunoreactivity in transverse nerves provided evidence for the release of MasITP from the transverse nerves into the hemolymph at insect ecdysis. These data suggest the presence of an insect ITP in M. sexta and a role for this hormone during ecdysis. This research was funded by the National Institutes of Health (MBRS SCORE Program-NIGMS) to M.F. (grant no. 2S06 GM52588-09), by the National Center on Minority Health and Health Disparities (grant no. 5P20-MD000262), an NIH RISE graduate fellowship to A.L.D. (5 R25 GM59298), an NIH PREP fellowship to C.C.H. and M.A.U. (5 R25 GM64078), an NSF CSU LS-AMP fellowship to C.C.H. (HRD-9802113), and by NIH MBRS-MARC to M.D.P. (T34 GM08574) and NIH MA/MS-PhD Bridge Scholarship to A.L.D. and C.C.H. (5R25 GM48972).     

Adaptive Evolution of the First Extra Exon in the Murid Rodent Prolactin Gene Family

The prolactin gene family in rodents consists of multiple members that coordinate the processes of reproduction and pregnancy. Some members of this family acquired one or two additional exons between exon 2 and exon 3 of the prototypical 5-exon, 4-intron structure, but the evolutionary importance of this insertion is unclear. Here, we focus on those members and survey this question by molecular evolutionary methods. Phylogenetic analysis shows that those members cluster into two distinct groups. Further analysis shows that the two groups of genes originated before the divergence of mouse and rat but after that of rodents from other mammals. We compared the d _N/d _S values for each branch of the gene tree but found no evidence to support positive selection for any branch. We found strong evidence, however, that one site (11E) of the 13 sites of the first extra exon underwent positive selection by the site-specific models of the maximum-likelihood method. Combining our molecular evolutionary analysis with other known functional evidence, we believe that the insertion of the extra exon implies some functional adaptation.     

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.     

Immunocytochemical identification of crustacean hyperglycemic hormone-producing cells in the brain of a freshwater fairy shrimp,Streptocephalus dichotomus Baird (Crustacea: Anostraca)

Localization of crustacean hyperglycemic hormone (CHH) activity in the brain ganglia of Streptocephalus dichotomus was demonstrated by immunocytochemical method. For this, two different rabbit antisera, one raised against a crayfish Orconectus limosus, and the other with a crab Carcinus maenas, were used. Positive immunoreactivity was recorded with the antibody raised against Orconectus limosus CHH. However, the antibody raised against the CHH of the crab, Carcinus maenas failed to show any cross-reactivity. The biological specificity of these peptides was further confirmed by immunoblotting and immunodiffusion studies performed with the partially purified CHH. The results obtained with the immunodot-blotting and immunodiffusion studies with CHH, further confirmed the immunocytochemical studies. Bioassay experiments performed with a freshwater prawn, Macrobrachium rosenbergii indicate the interspecific biological activity of this neuropeptide. The present study indicates that besides the sinus gland, there are other sites of CHH synthesis and release in the brain ganglia of S. dichotomus.     

miR‐34 regulates reproduction by inhibiting the expression of MIH,CHH, EcR,and FAMeT genes in mud crab Scylla paramamosain

Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR‐34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR‐34 in eyestalk. Six reproduction‐related genes with an intact 3′‐untranslated region (UTR), including molt‐inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis‐inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3′‐UTR plasmid vectors of the six genes were cotransfected with miR‐34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR‐34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR‐34 and antagomiR‐34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real‐time polymerase chain reaction. The results showed that agomiR‐34 suppressed the expression of the four genes, whereas antagomiR‐34 enhanced their expression. These experimental results confirmed our hypothesis that miR‐34 may indirectly regulate reproduction via binding to the 3′‐UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.     

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.     

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.     

Do the neurohormones VIH (vitellogenesis inhibiting hormone) and CHH (crustacean hyperglycemic hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster,Homarus americanus

Summary

The present study deals with the location of the vitellogenesis inhibiting hormone (VIH)-producing cells in the eyestalk of the lobster Homarus americanus. In the present study, the neurosecretory pathways of VIH in Homarus, have been described immunocytochemically by use of a mouse serum against Homarus VIH. The location of the VIH cells was compared with the location of the crustacean hyperglycemic hormone (CHH) cells visualized by a rabbit serum raised against CHH of the crayfish Astacus leptodactylus. Immunocytochemical detection procedures, both at the light and electron microscopic level, revealed frequent but not complete co-localization of VIH and CHH in a variable number of the same group of perikarya. In the sinus gland, both neuropeptides were mostly demonstrated in distinct axonal endings characterized by different granule types. Postulations on the biosynthesis of these factors and suggestions concerning the processing of both neurohormones have been made.     

西葫芦NCED基因家族鉴定及其响应干旱胁迫分析

NCED基因家族成员在调节植物响应干旱胁迫中发挥着关键作用,该研究通过生物信息学技术分析NCED在西葫芦基因组中的分布、结构及进化,研究家族成员在不同组织中的表达特异性及其对10%PEG 6000模拟干旱、0.1 mmol·L-1ABA激素和自然干旱胁迫的响应,以解析NCED基因家族的生物学功能。结果表明:(1)从西葫芦全基因组中鉴定出6个NCED家族基因(CpNCED1~6),且6个基因均不含内含子、分别分布于西葫芦的1、10、12、14、19和20号共6条染色体上。(2)理化性质分析发现,CpNCED1~6蛋白长度为569~590 aa,理论分子量在62.64~65.54 kD之间。(3)蛋白保守元件分析显示,除CpNCED3蛋白在遗传进化过程中出现3个基序(motif 12、motif 13和motif 15)的缺失外,其余5个蛋白都有完整的16个motif保守基序,且分布在600个氨基酸以内,同时大部分NCED蛋白序列保守性较高。(4)顺式作用元件分析显示,西葫芦CpNCED1~6基因均含ABRE、W box、MBS、P-box、TCA-element、CGTCA-motif、TGA-element和TGA-box等潜在的干旱胁迫响应元件。(5)qRT-PCR分析表明,CpNCED1~6基因在西葫芦不同组织中的表达具有组织特异性,其中,CpNCED4和CpNCED1在茎中的表达量显著高于其他4个基因,CpNCED2、CpNCED4、CpNCED6在花中的表达显著高于其余3个基因且CpNCED2表达量最高,CpNCED1~6在果实和叶中的表达量均相对较低;与对照组相比,CpNCED1~6受模拟干旱、ABA激素和自然干旱胁迫均上调表达;伴随干旱胁迫的产生,叶片中脱落酸(ABA)含量逐渐升高,暗示CpNCEDs在西葫芦干旱胁迫响应与ABA的生物合成过程中发挥着正向调控作用。研究发现,6个CpNCED1~6基因与西葫芦干旱胁迫响应密切相关,且对西葫芦干旱胁迫的响应以及ABA生物合成具有重要作用,尤其以CpNCED2和CpNCED4基因的作用更为明显。