Recombinant Crustacean Hyperglycemic Hormone Research Articles

Metabolic and osmoionic effects of the recombinant crustacean hyperglycemic hormone (rCHH-A) of the Pacific white shrimp Penaeus vannamei on specimens acutely exposed to extreme salinities

The crustacean hyperglycemic hormone (CHH) is a multifunctional neuropeptide that plays a central role in crustacean metabolism and physiology. Experiments were conducted to examine the metabolic and osmoionic capabilities of the white shrimp Penaeus vannamei injected with its recombinant CHH-A (rCHH-A) peptide and acutely transferred from iso- (26 ppt) to hypo- (10 ppt) and hyper-osmotic (40 ppt) conditions. Hemolymph glucose, protein, osmoregulatory capacity (OC), and ionoregulatory capacity (IC) for sodium, chloride, potassium, and calcium were evaluated at four post-injection times (0.5, 1, 2, and 24 h). The rCHH-A peptide had hyperglycemic activity in all salinity conditions, obtaining maximum values at 1 h post-injection. However, in shrimp transferred to hyper-osmotic condition, rCHH-A caused the most significant reductions in OC (2 h), chloride IC (2 h), and total proteins (0.5-2 h) compared to the phosphate-buffered saline (PBS) control. Contrastingly, in shrimp transferred to hypo-osmotic conditions, rCHH-A decreased OC significantly from 2-24 h, strongly decreased chloride and potassium IC at 1 h post-injection, and increased total protein concentration in the hemolymph (1-2 h) when compared to PBS control. Concerning calcium, the rCHH-A injection decreased calcium IC at 10 ppt (1 h) and 26 ppt (2 h), providing insight into a potential role for CHH-A in calcium regulation. The results suggest that glucose and protein mobilization could enhance energy for osmo-ionic regulation under extreme osmotic conditions. This research study contributed to understanding crustacean endocrinology in P. vannamei and related euryhaline crustaceans. Further research should be performed to understand the osmo- and ionoregulatory mechanisms of the different CHH variants in crustaceans exposed to other stress conditions and the relationship with intermediary energy metabolism regulation.

Cloning and expression of the recombinant crustacean hyperglycemic hormone isoform B2 (rCHH-B2) and its effects on the metabolism and osmoregulation of the Pacific white shrimp Litopenaeus vannamei

Crustacean hyperglycemic hormones (CHHs) are multifunctional neuropeptides ubiquitous in crustaceans. In Litopenaeus vannamei, CHH-B2 is a CHH eyestalk isoform whose expression has been shown to vary with enviromental conditions, suggesting its relevance for ecophysiological performance of shrimp, controlling processes related to metabolism and osmo-ionic regulation. To study the involvement of CHH-B2 in these processes, we cloned and expressed a recombinant version with a free C-terminal glycine (rCHH-B2-Gly) in the methylotrophic yeast Pichia pastoris. The rCHH-B2-Gly peptide secreted to the culture medium was purified by RP-HPLC and used for in vivo glucose, triglyceride, and osmoregulation dose-response analyses with juvenile shrimp. The peptide was also amidated at the C-terminus using an α-amidating enzyme to produce rCHH-B2-amide. The shrimp showed a dose-dependent effect of rCHH-B2-Gly to hemolymph glucose and triglyceride levels, inducing maximal increases by injecting 500 and 1000pmol of hormone, respectively. Additionally, 10pmol of hormone was sufficient to reduce the hypo-osmoregulatory capacity of shrimp at 35‰. These findings suggest that CHH-B2 has regulatory roles in carbohydrate and lipid metabolism, and a potential involvement in osmoregulation of L. vannamei. Injection of 100pmol of rCHH-B2-amide increased glucose and triglyceride levels by 15 and 28%, respectively in comparison with rCHH-B2-Gly, suggesting an important role for the C-terminal amidation. Additionally, an in silico structural analysis done with the CHH-B1 and rCHH-B2-Gly peptides suggests that the C-terminal region may be relevant for the activity of the L. vannamei isoforms and explain the functional divergence from other crustacean CHH/CHH-like peptides.

Involvement of the crustacean hyperglycemic hormone (CHH) in the physiological compensation of the freshwater crayfish Cherax quadricarinatus to low temperature and high salinity stress.

This study was aimed at determining the role of the crustacean hyperglycemic hormone (CHH) in the physiological compensation to both saline and thermal stress, in the freshwater crayfish Cherax quadricarinatus. By determining the expression of the CHH gene in the eyestalk of juvenile crayfish, we found that maximal induction of CHH was induced at high salinity (10 g/L) and low temperature (20 °C). In order to investigate the role of CHH in the physiological compensation to such stressful conditions, recombinant CHH was supplied to stressed animals. CHH-injected crayfish showed increased hemolymphatic levels of glucose, in accordance with a significant utilization of glycogen reserves from the hepatopancreas. Furthermore, CHH administration allowed stressed animals to regulate hemolymphatic sodium and potassium at more constant levels than controls. Taken together, these results suggest a relevant role of CHH in increasing the energy available intended for processes involved in the physiological compensation of C. quadricarinatus to both saline and thermal stress.

A chimeric recombinant crustacean hyperglycemic hormone fromLitopenaeus schmitti(Burkenroad) obtained as C-terminus fusion protein boost hemolymph glucose concentration inLitopenaeus vannamei(Boone)

To date, no hormonal treatments are available for control of shrimp reproduction and only eyestalk ablation is of practical use. The crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide of the eyestalk CHH family. It plays an important role in the regulation of hemolymph glucose levels, as its principal function, but it is also implicated in additional physiological processes such as moulting and reproduction. In the present study, the cDNA encoding Litopenaeus schmitti (Burkenroad) mature CHH was cloned into the Escherichia coli pTYB2 expression vector. Using this strategy we have obtained, for the first time, the recombinant CHH from L. schmitti with its C-terminus fused to an intein tag. The expected fused protein of about 63 KDa was expressed in E. coli forming inclusion bodies. It was purified in a soluble form by electroelution following molecular size fractionation in sodium dodecil sulphate polyacrylamide gel electrophoresis. The ability of the chimeric CHH protein to elevate glucose levels in the hemolymph of the eyestalk-ablated Litopenaeus vannamei (Boone) shrimps indicates that its biological activity as hyperglycemic protein is preserved. The results provide an alternative tool to obtain soluble recombinant proteins from the CHH family of neuropeptides to get a better understanding of shrimp endocrinology.

Effects of crustacean hyperglycemic hormone (CHH) on the transcript expression of carbohydrate metabolism-related enzyme genes in the kuruma prawn, Marsupenaeus japonicus

Crustacean hyperglycemic hormone (CHH), a member of a neuropeptide family present only in arthropods, plays a pivotal role in the modulation of hemolymph glucose levels, molting, reproduction, and the stress response. Although it has been determined that hepatopancreas and muscle are the major tissues in which CHH regulates hyperglycemic activity, the molecular mechanism by which CHH regulates carbohydrate metabolism remains unclear. In this study, we analyzed the mRNA expression levels of enzymes involved in glycogen metabolism and gluconeogenesis in order to determine how CHH regulates hemolymph glucose levels. We first cloned cDNAs encoding four carbohydrate metabolism-related enzymes from the kuruma prawn, Marsupenaeus japonicus, glycogen phosphorylase (MjGP), glycogen synthase (MjGS), fructose 1,6-bisphosphatase (MjFBPase), and phosphoenolpyruvate carboxykinase (MjPEPCK). RT-PCR analysis showed that eyestalk ablation remarkably decreased MjGP and increased MjGS transcript levels in the hepatopancreas, but not in muscle. Considering the fact that various eyestalk factors, including MIH, are removed by eyestalk ablation, these results indicate that after eyestalk ablation the metabolic state proceeds towards glycogen accumulation in the specific tissues related to molting. In contrast, MjFBPase and MjPEPCK transcript levels were not significantly changed by eyestalk ablation, indicating that CHH and other eyestalk-derived factors might not induce gluconeogenesis. Quantitative real-time PCR analysis showed that exposure of hepatopancreas to recombinant CHH significantly changed the expression levels of MjGP and MjGS, but not MjFBPase and MjPEPCK. Collectively, these results indicate that CHH is involved in glycogen metabolism in hepatopancreas.

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp ( Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp ( Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1 h after the rLV-CHH injection and then returned to normal after 3 h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.

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 α-helices, and are similarly thermo-stable with a melting temperature of 74–75 °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 α-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 α-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 α-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.

Identification of a Second Messenger of Crustacean Hyperglycemic Hormone Signaling Pathway in the Kuruma Prawn Marsupenaeus japonicus

To address the signaling pathway of crustacean hyperglycemic hormone (CHH) in the kuruma prawn Marsupenaeus japonicus, we measured the intracellular cAMP and cGMP levels in the hepatopancreas, one of the major target tissues of CHH. Removal of CHH-producing cells by bilateral eyestalk ablation decreased intracellular cGMP levels, while little effect on cAMP was observed in the tissue. Similarly, exposure to recombinant CHH significantly increased the cGMP level of the hepatopancreas in vitro, while little change was observed in cAMP. These results suggest that cGMP, rather than cAMP, mediates the CHH signal in hepatopancreas of M. japonicus.

A convenient method for preparation of biologically active recombinant CHH of the kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.

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.