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.     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

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.     

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.     

Antipeptide antibodies for detecting crab (Callinectes sapidus) molt-inhibiting hormone

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.     

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase beta-subunit C-terminal mutants.

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.     

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.     

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%.     

中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达

根据实验室分离自中华绒螯蟹(Eriocheir sinensis)的一种蜕皮抑制激素(Molting-inhibiting hormone,MIH)N端氨基酸测序结果设计简并引物,采用RACE方法,首次从中华绒螯蟹眼柄中克隆到蜕皮抑制激素基因全长cDNA(Es-MIH,GenBank登录号:DQ341280),该基因全长为1457 bp,开放阅读框为330 bp,编码110个氨基酸(含有35个氨基酸的信号肽);其成熟肽包含C7-C44、C24-C40和C27-C53三个二硫键,有典型的CHH家族结构域。该cDNA编码的氨基酸序列与地蟹(Gecarcinus lateralis)MIH同源性最高,达到了85%。Northern杂交和半定量RT-PCR显示蜕皮间期成体蟹仅在眼柄中有MIH基因表达,提示该基因的表达具有一定组织特异性。利用pCR T7/NT TOPO TA系统重组表达MIH成熟肽,纯化的重组蛋白得率为0.3 g/L,纯化产物经质谱鉴定为中华绒螯蟹MIH。研究解决了CHH家族神经肽在机体中的表达量少,直接纯化较难的问题,为深入研究MIH的作用机制和在生产上控制中华绒螯蟹蜕皮和生长奠定了基础。     

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.     

Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.     

In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus

In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0 +/-0.4 days in the control group, 9.5+/-0.5 days in the 2500 ng/g-body weight/injection-group, mean+/-SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94+/-1.09 in the control and 1.28+/-0.39 in the 3000 ng/g-body weight/injection-group, mean+/-SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.     

Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.     

Expression of a recombinant molt-inhibiting hormone of the kuruma prawn Penaeus japonicus in Escherichia coli.

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.     

Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.     

Interacting Factors in the Control of the Crustacean Molt Cycle

In order to account for the known phenomena of the crustaceanmolt cycle, at least six factors must be postulated: a moltinghormone (20-OH-ecdysone), a molt-inhibiting hormone (MIH), ananecdysial limb autotomy factor, a proecdysial limb-autotomyfactor, a limb growth-inhibiting factor and an exuviation factor.Only the molting hormone and its derivatives have been chemicallywell defined. The various factors interact in complex ways tomaintain not only a coordinated proecdysial period in preparationfor exuviation but also a proecdysial period with the flexibilityto respond to such interim hazards as the loss of partiallyregenerated limbs.     

Molecular mechanisms regulating molting in a crustacean

Crustacean growth and development is characterized by periodic shedding (ecdysis) and replacement of the rigid exoskeleton. Secretions of the X-organ sinus gland complex control the cellular events that lead to growth and molting. Western blot and ELISA results showed a progressive increase in growth arrest-specific protein (Gas7) from early postmolt stage to a maximum at late postmolt stage. Phosphorylation of ERK2, a downstream signaling protein, was also identified in the subsequent stages. ERK2 phosphorylation resulted in the expression of molt-inhibiting hormone (MIH). Specific ERK inhibitors (PD98059 and UO126) exhibited the ability to reduce the molting duration of Fenneropenaeus indicus from 12-14 days to 7-8 days, suggesting that the ERK1/2 signaling pathway is responsible for the expression of MIH, which controls the molt cycle. We have identified the stage-specific expression of Gas7 (approximately 48 kDa) in the X-organ sinus gland complex of eyestalk which is involved in the downstream signaling of the ERK1/2 pathway regulating the expression of MIH during the molt cycle of the white shrimp, F. indicus. These are the first data showing an association between the Gas7 signal-transduction process and regulation of the molt cycle and provides an alternative molecular intervention mechanism to the traditional eyestalk ablation in crustaceans.