Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones from the lobster Homarus americanus

Using the polymerase chain reaction with degenerated oligonucleotides, we have isolated cDNA clones that encode two structurally different (92% identity) crustacean hyperglycemic hormones (CHH) from the lobster Homarus americanus. The deduced amino acid sequences fully agree with previously published data on partial amino acid sequences, amino acid compositions and molecular masses of hyperglycemic peptides in the lobster. A comparative analysis between the deduced primary structure of two lobster CHH and the crab CHH sequence reveals a phylogenetic relationship and allows the prediction of biologically important regions within the structures of these novel neuropeptides.     

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.     

Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.     

Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster,Jasus lalandii

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.     

Endocrine cells in the gut of the shore crab Carcinus maenas immunoreactive to crustacean hyperglycaemic hormone and its precursor-related peptide

The distribution and morphology of gut endocrine cells, which are immunoreactive to crustacean hyperglycaemic hormone (CHH) and the corresponding precursor-related peptide (CPRP), have been described in the shore crab Carcinus maenas. The cells are uniquely distributed throughout the fore- and hindgut, but were never observed in the midgut or associated caeca. Expression of CHH and CPRP in the gut endocrine cells is generally restricted to premoult, although small numbers of immunoreactive cells were observed in intermoult and postmoult. A notable feature of the distribution of these slender cells was that, whilst they are distributed evenly over much of the fore- and hindgut, all extrinsic and intrinsic muscles of the gastric and pyloric stomach examined were surrounded by a ring(s) of cells, suggesting a mechanoreceptive function. Ultrastructural studies revealed that these cells contain numerous immunopositive, electron-dense granules. This suggests that they are "paraneurones", which secrete CHH and CPRP into the haemolymph during ecdysis, accounting for the ecdysial surge in CHH, which is implicated in water uptake and swelling prior to ecdysis.     

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.     

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.     

Positive Darwinian selection on crustacean hyperglycemic hormone (CHH) of the green shore crab, Carcinus maenas

The tissue-specific expression and differential function of the crustacean hyperglycemic hormone (CHH) in Carcinus maenas indicate an interesting evolutionary history. Previous studies have shown that CHH from the sinus gland X-organ (XO-type) has hyperglycemic activity, whereas the CHH from the pericardial organ (PO-type) neither shows hyperglycemic activity nor it inhibits Y-organ ecdysteroid synthesis. Here we examined the types of selective pressures operating on the variants of CHH in Carcinus maenas. Maximum likelihood-based codon substitution analyses revealed that the variants of this neuropeptide in C. maenas have been subjected to positive Darwinian selection indicating adaptive evolution and functional divergence among the CHH variants leading to two unique groups (PO and XO-type). Although the average ratio of nonsynonymous to synonymous substitution (omega) for the entire coding region is 0.5096, few codon sites showed significantly higher omega (10.95). Comparison of models that incorporate positive selection (omega > 1) with models not incorporating positive selection (omega <1) at certain codon sites failed to reject (p=0) evidence of positive Darwinian selection.     

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.     

Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus

Crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs) are produced during the proteolytic processing of CHH preprohormones. Currently, the physiological roles played by CPRPs are unknown. Due to their large size, direct mass spectrometric sequencing of intact CPRPs is difficult. Here, we describe a novel strategy for sequencing Cancer productus CPRPs directly from a tissue extract using nanoflow liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Four novel CPRPs were characterized with the aid of MS/MS de novo sequencing of 27 truncated CPRP peptides. Extensive modifications (methionine oxidation and carboxy-terminal methylation) were identified in both the full-length and truncated peptides. To investigate the origin of the modifications and truncations, a full-length CPRP was synthesized and subjected to the same storage and extraction protocols used for the characterization of the native peptides. Here, some methionine oxidation was seen, however, no methylation or truncation was evident suggesting much of the chemical complexity seen in the native CPRPs is unlikely due to a sample preparation artifact. Collectively, our study represents the most complete characterization of CPRPs to date and provides a foundation for future investigation of CPRP function in C. productus.     

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.     

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.     

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides

To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.     

Amino acid sequence of putative moult-inhibiting hormone from the crab Carcinus maenas.

Putative moult-inhibiting hormone (MIH) was isolated from sinus glands of the shore crab Carcinus maenas, and its primary structure determined by automated Edman degradation of endoproteinase derived peptide fragments. MIH is a 78 residue neuropeptide (deduced molecular mass 9181 Da) with three disulphide bridges and unblocked N- and C-termini. MIH shows some homology to the crustacean hyperglycemic hormone (CHH) neuropeptide family. However, consideration of the roles of various members of this group, together with sequence information recently reported, strongly suggests that these neuropeptides may be multifunctional.     

A common precursor to two major crab neurosecretory peptides

The biosynthesis of proteins by the X-organ sinus gland (XOSG) neurosecretory system of the crab, Cardisoma carnifex was studied using the pulse-chase technique. Analysis of radioactive proteins following 2D-PAGE showed that during pulse incubations of less than or equal to 30 min a single predominant 14Kd prohormone was synthesized. With chase less than or equal to 3 hr the primary 14Kd protein was found to undergo differential and/or multiple post-translational modifications prior to its proteolytic cleavage. Increasing the chase to greater than 3 hr showed a shift in labeling from the 14Kd forms to 3 separate 6Kd proteins. Two of the 6Kd proteins were identified as crustacean hyperglycemic peptides (CHH). Similarity in protein labeling using [3H]leucine and [35S]cysteine suggest a second major peptide group, the H peptide, known to lack cysteine, is also contained within the 14Kd precursor. Peptide mapping of the 14Kd proteins and of unlabeled CHH and peptide H provide substantive evidence for this biosynthetic scheme. Thus, both the CHH and H peptide groups, which together constitute greater than 90% of the XOSG peptide content, in this species, arise from a common 14Kd precursor molecule.     

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Primary structure of CHH/MIH/GIH-like peptides in sinus gland extracts from Penaeus vannamei

Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7-9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.     

Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity

Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.