Chi-Ying Lee

Crustacean molt-inhibiting hormone: Structure, function, and cellular mode of action

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.

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.

Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea.

Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further alpha-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower alpha-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 alpha-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.

Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes

Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7 fmol/mg protein) and SGCs (497.8±49.4 fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.

Neuroendocrine responses of a crustacean host to viral infection: Effects of infection of white spot syndrome virus on the expression and release of crustacean hyperglycemic hormone in the crayfish Procambarus clarkii

The objectives of the present study were to characterize the changes in crustacean hyperglycemic hormone (CHH) transcript and peptide levels in response to infection of white spot syndrome virus (WSSV) in a crustacean, Procambarus clarkii. After viral challenge, significant increase in virus load began at 24 h post injection (hpi) and the increase was much more substantial at 48 and 72 hpi. The hemolymph CHH levels rapidly increased after viral challenge; the increase started as early as 3 hpi and lasted for at least 2 d after the challenge. In contrast, the hemolymph glucose levels did not significantly changed over a 2 d period in the WSSV-infected animals. The CHH transcript and peptide levels in tissues were also determined. The CHH transcript levels in the eyestalk ganglia (the major site of CHH synthesis) of the virus-infected animals did not significantly change over a 2 d period and those in 2 extra-eyestalk tissues (the thoracic ganglia and cerebral ganglia) significantly increased at 24 and 48 hpi. The CHH peptide levels in the eyestalk ganglia of the virus-infected animals significantly decreased at 24 and 48 hpi and those in the thoracic ganglia and cerebral ganglia remained unchanged over a 2 d period. These data demonstrated a WSSV-induced increase in the release of CHH into hemolymph that is rapid in onset and lasting in duration. Changes in the CHH transcript and peptide levels implied that the WSSV-induced increase in hemolymph CHH levels primarily resulted from an enhanced release from the eyestalk ganglia, but the contribution of the 2 extra-eyestalk tissues to hemolymph pool of CHH increased as viral infection progressed. The combined patterns of change in the hemolymph glucose and CHH levels further suggest that the virus-enhanced CHH release would lead to higher glycolytic activity and elevated glucose mobilization presumably favorable for viral replication.

Characterization of the neuropeptidome of a Southern Ocean decapod, the Antarctic shrimp Chorismus antarcticus: Focusing on a new decapod ITP-like peptide belonging to the CHH peptide family

As part of the study of the resilience of Antarctic crustaceans to global warming, the shrimp Chorismus antarcticus was subjected to an analysis of global approach using the Next Generation Sequencing Illumina Hi-Seq platform. With this data a detailed study into the principal neuropeptides and neurohormones of this species have been undertaken. Total RNAs from whole animals were enriched with eyestalk extracts to ensure maximum sequencing depth of the different neurohormones and neuropeptides mainly expressed into the X organ-sinus gland complex, which is a major endocrine organ of their synthesis. Apart from the information that can provide the availability of the transcriptome of a polar crustacean, the study of neuropeptides of a caridean shrimp will partially fill the limited data available for this taxon. Illumina sequencing was used to produce a transcriptome of the polar shrimp. Analysis of the Trinity assembled contigs produced 55 pre-pro-peptides, coding for 111 neuropeptides belonging to the following families: adipokinetic-corazonin-like peptide, Allatostatins (A, B et C), Bursicon (α), CCHamide, Crustacean Hyperglycemic Hormones (CHH), Crustacean Cardioactive Peptide (CCAP), Corazonin, Crustacean Female Sex Hormone (CSFH), Diuretic Hormones 31 and 45 (DH), Eclosion Hormone (EH), FLRFamide, GSEFLamide, Intocin, Ion Transport Peptide-like (ITP-like), Leucokinin, Molt-inhibiting Hormone, Myosuppresin, Neuroparsin, Neuropeptide F (NPF), Orcokinin, Orcomyotropin, Pigment Dispersing Hormone (PDH), Pyrokinin, Red Pigment Concentrating Hormone (RPCH), SIFamide, small Neuropeptide F (sNPF), Sulfakinin and finally Tachykinin Related peptides. Among the new peptides highlighted in this study, the focus was placed on the peptides of the CHH family and more particularly on a new ITP-like in order to confirm its belonging to a new group of peptides of the family. A phylogeny made from more than 200 sequences of peptides, included new sequences from new species besides Chorismus antarcticus, confirms the peculiarity of this new set of peptides gathered under the name ITP-like.

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.

Metabolic effects of parasitization by the barnacle Polyascus plana (Cirripedia: Rhizocephala: Sacculinidae) on a grapsid host, Metopograpsus thukuhar

Pathophysiological studies of rhizocephalan infections are rare. We describe differences in the levels of tissue and hemolymph metabolites between Polyascus plana-parasitized and unparasitized individuals of Metopograpsus thukuhar. Crabs were assigned to either a parasitized (carrying at least 1 externa, i.e. a protruding reproductive body) or an unparasitized (not carrying externae and determined to be rootlet-free by a barnacle 18S rRNA-based polymerase chain reaction) group. Quantification of metabolites showed that muscle glycogen levels were significantly lower and hepatopancreas levels were significantly higher in parasitized crabs compared to unparasitized crabs; hepatopancreas triacylglycerol levels were significantly higher and hemolymph levels significantly lower in parasitized hosts, and there was no significant difference in muscle triacylglycerol levels between unparasitized and parasitized animals. Glucose levels in the hepatopancreas, muscle, and hemolymph were all significantly higher in parasitized hosts. Significant levels of glucose, triacylglycerol, and glycogen were present in the barnacle externae. In addition, levels of crustacean hyperglycemic hormone in the sinus glands were not significantly different between unparasitized and parasitized animals. Glucose mobilized from the muscle is likely converted to glycogen and triacylglycerol in the rootlet-infiltrated hepatopancreas of parasitized hosts, and the eyestalk neuroendocrine system appears not to be significantly impaired, in terms of hormone production and storage, by parasitization.