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.     

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.     

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the -factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.     

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 biology of neurohormone precursors in the eyestalk of Crustacea

Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.     

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.     

Secretion of Pem-CMG,a peptide in the CHH/MIH/GIH family of Penaeus monodon,in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.     

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ecdysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CHH/MIH/GIH family. The cloning of MIH (formerly MIH-like) of the shrimp Metapenaeus ensis has been reported in a previous study. To obtain a large quantity of fusion protein for antibody production and biological assay, the cDNA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-charged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Western blot analysis indicated that the antibody was specific. Furthermore, results from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH, shrimp at intermolt stage were injected with rMIH and the molt cycle duration of the injected shrimp was monitored. A significant increase in molt cycle duration was recorded for the shrimp injected with the recombinant protein.     

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

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

Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans

Background  

Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated.     

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.     

中华绒螯蟹蜕皮抑制激素基因的表达及抗体制备

蜕皮抑制激素(Moltinhibitinghormone,MIH)属于甲壳动物高血糖激素家族神经肽,对甲壳类的蜕皮起抑制作用。本研究用DNA重组技术将中华绒螯蟹(Eriocheirjaponicasinensis)的蜕皮抑制激素1(ErsMIH1)成熟肽的cDNA序列亚克隆至原核表达载体pET28a( )中,并在大肠杆菌BL21(DE3)中进行高效表达。SDSPAGE检测结果显示,融合蛋白pET-MIH1的Mr约为12kD,与理论值相符。融合蛋白的表达量约占菌体总蛋白的15%,表达产物以包涵体形式存在。对包涵体进行变性、复性及纯化处理,并以8mol/L尿素溶解的包涵体作为免疫原免疫BALB/c小鼠制备多克隆抗体。ELISA和Westernblot的结果表明制备的抗体效价高、特异性强     

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.