The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare.

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C-peptide (50 amino acids). These results were confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex-determination. In invertebrates, there is no other example where sex-differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti-Müllerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.     

Immunological identification of crustacean androgenic gland hormone, a glycopeptide

Androgenic gland hormone (AGH) is known to be responsible for sex differentiation in crustaceans. The amino acid sequence of AGH-active fraction purified from androgenic glands of the terrestrial isopod Armadillidium vulgare was determined by immunoprecipitation employing three types of antibodies raised against differing parts of the amino acid sequence deduced from the putative AGH cDNA sequence. As all antibodies adsorbed AGH activity, it was confirmed that the sequence examined was that of AGH. The affinity of AGH to certain lectins indicated that AGH possesses a carbohydrate moiety, which is in agreement with the observation that AGH possesses an N-glycosylation consensus sequence.     

Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.     

几种甲壳动物促雄性腺素结构预测分析

利用生物信息学方法对目前已知的3种甲壳动物促雄性腺素前体(AGH precursor)和5种类胰岛素促雄性腺因子(insulin-like AG factor)进行分析,探讨了促雄性腺素前体的氨基酸理化特性、信号肽、跨膜结构域、二级结构、motif等,并利用Phyre软件对其三级进行同源性收索。结果显示:促雄性腺素前体包含信号肽,存在跨膜结构域,并和信号肽同位。PDB库中没有找到匹配的motif。3种促雄性腺素前体的二级结构有比较高的相似性,比如都包含两个中心螺旋区。Phyre搜索显示,与8种蛋白的三级结构匹配的均为胰岛素家族的蛋白,这也进一步证实了促雄性腺素前体和胰岛素原的相似性。     

Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): Total chemical synthesis of IAG

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.     

Molecular evolution of the androgenic hormone in terrestrial isopods

In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts.     

The primary structure and the disulfide links of the bovine relaxin-like factor (RLF).

The relaxin-like factor (RLF), produced by the Leydig cells, is an essential link in the chain of events leading to the proper positioning of the testes during fetal development. The primary structure of RLF, as reported in the literature, is based solely upon cDNA sequences with chain lengths determined according to deduced processing sites and with relaxin-like cross-links. Biochemical characterization of bovine testicular RLF shows clearly that the endogenous hormone does consist of a 26 residue A chain and two forms of B chain, one containing 40 residues, the other 45. In addition, both B chains are 9 residues longer at the C terminus than the cDNA-deduced chain, and about 20% of the B chains have an additional 5 residue extension at the N terminus. Sequence analysis in combination with mass spectrometry and tryptic peptide mapping showed unambiguously that RLF is larger than previously assumed and that it has the relaxin-type disulfide bond distribution that makes it a bona fide member of the relaxin family of hormones.     

Hormonal Control of Sexual Differentiation and Reproduction in Crustacea

SYNOPSIS. Sexual differentiation in malacostracan Crustaceais controlled by the androgenic gland hormone (AGH). In males,the primordial androgenic glands (AG) develop and AGH inducesmale morphogenesis. In females, the primordial AG does not developand the ovaries differentiate spontaneously. Implantation ofthe AG into females yields various results, showing that thesensitivity to AGH differs with the species and the receptiveorgans. Purified AGH of the isopod Armadillidium vulgare consistsof at least two molecular forms, which exist as monomeric proteinswith molecular weights of 17,000 ± 800 and 18,300 ±1,000 Da and with isoelectric points of about 4.5 and 4.3, respectively.The antiserum raised against purified AGH makes it possibleto measure AGH activity by immunoassay. Neurohormones control male and female reproduction. In males,they are involved in the maintenance of the male germinativezone and the control of AG activity. In females, the secondaryvitellogenesis is controlled by the vitellogenesis-inhibitinghormone (VIH) and the vitellogenesis- stimulating hormone (VSH).VIH isolated from the lobster Homarus americanus is a peptidewith a molecular weight of 9,135 Da and shows homology to thecrustacean hyperglycemic hormone and moltinhibiting hormone.Involvement of the molting hormone and the juvenile hormone-likecompound in the secondary vitellogenesis have also been suggested.In the amphipod Orchestia gammarella, the vitellogenesis- stimulatingovarian hormone (VSOH) seems to control vitellogenin synthesis     

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

The structural basis of the multiple forms of human complement component C4

cDNA clones of human complement components C4A and C4B alleles were prepared from mRNA obtained from the liver of a donor heterozygous at both loci. cDNA from one C4A allele was sequenced to give the derived complete amino acid sequence of 1722 amino acid residues of the C4 single chain precursor molecule and the estimated sequences of the three peptide chains of secreted C4. Comparison with partial sequences of a second C4A allele and a C4B allele has led to the tentative identification of some class differences in nucleotide sequences between C4A and C4B and of allelic differences between C4A alleles in this highly polymorphic system.     

Endocrine and genetic control of sex differentiation in the Malacostracan Crustacea

Summary

Sex differentiation in Malacostraca is controlled by hormone secreted from the androgenic glands. Experimentally induced sex inversions in isopods and amphipods proved that the genetic female and male possess primordia of the androgenic glands, gonads, and gonoducts, along with sexual characteristics of both sexes. During the sensitive period, the presence or absence of androgenic gland hormone (AGH) affects the differentiation of these primordia.

Genetic control of the development of androgenic gland primordium seems to be brought about assuming of the following: 1. Both genetic female and male possess gene(s) (AGH-G) responsible for the AGH-synthesis situated on the homologous loci of the sex chromosomes and/or on the autosomes. 2. The gene(s) are activated spontaneously with the lack of inhibition of the major sex factor carried by the W or X chromosome. The W and X factors are hypostatic to major sex factor carried by the Y chromosome. The Z factor does not seem to influence sex differentiation. Sufficient allochthonous AGH seems to render the W and X factors ineffective.     

Nucleotide sequence of cloned cDNA coding for preproricin

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).     

革胡子鲶生长激素cDNA克隆与蛋白质结构分析

从革胡子鲶(Clarias lazera(Burchell))的脑垂体组织中提取总RNA, 应用RT-PCR方法, 扩增得到了革胡子鲶生长激素(Growth hormone, GH)基因cDNA的开放阅读框(Open reading frame, ORF)序列。ORF全长为603 nt, 编码由22个信号肽氨基酸和178个成熟肽氨基酸共同组成的生长激素前体蛋白。序列同源比较结果表明, 研究中得到的革胡子鲶生长激素氨基酸序列与GenBank中已报道的其他6种鲶形目鱼类的氨基酸序列同源性高达95.8%。二级结构预测分析结果表明, 革胡子鲶生长激素蛋白中含有a 螺旋、b 折叠和b 转角以及无规卷曲等二级结构, 以a 螺旋为主, 是典型的a 型结构蛋白质。此外, 抗原性分析表明, 在氨基酸序列中的4个区域均可形成优势抗原表位, 其结构特点非常适合改造成为重组生长激素疫苗或单克隆抗体制剂加以开发利用。     

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.     

Use of the polymerase chain reaction to clone and sequence a cDNA encoding the bovine alpha 3 chain of type IV collagen

A novel type IV collagen, alpha 3(IV), has previously been isolated from a collagenase digest of bovine and human glomerular and lens basement membranes. The cloning and sequencing of a cDNA encoding the alpha 3(IV) chain is described here. Using the polymerase chain reaction, with primers derived from the known 27-residue bovine alpha 3(IV) amino acid sequence, a 68-base pair bovine genomic fragment (KEM68) which encodes the known peptide sequence, was synthesized. KEM68 was then used to screen a bovine lens cDNA library and a 1.5-kilobase partial cDNA clone obtained, encoding 471 residues of the bovine alpha 3(IV) chain: 238 residues from the triple helical collagenous domain and all 233 residues of the noncollagenous domain. The collagenous repeat sequence has three interruptions, coinciding with those in the alpha 1(IV) chain. The noncollagenous domain has 12 cysteine residues in identical positions to those of other type IV collagens and 71, 61, and 70% overall similarity with the human alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains. The noncollagenous domain of alpha 3(IV) is of particular interest as it appears to be the component of glomerular basement membrane that reacts maximally with the Goodpasture antibody. Furthermore, such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients with Alport syndrome. The alpha 3(IV) cDNA clone described here now permits study of the molecular pathology of COL4A3 in Alport syndrome.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

The Crustacean Androgen: A Hormone in an Isopod and Androgenic Activity in Decapods

The androgenic gland has been described in a variety of crustaceanspecies—isopods, amphipods and decapods. It has been shownto play a role in the regulation of male differentiation andin the inhibition of female differentiation. Upon its applicationfor endocrine manipulation, it inhibits female characteristics.Recently, the androgenic hormone from the isopod Armadillidiumvulgare was purified and characterized on the basis of a morphologicalbioassay. The hormone is a glycosylated protein composed oftwo peptide chains connected each to the other by two disulfidebridges. The pro-hormone consists of the same two chains connectedby a third peptide in a complex that resembles the insulin superfamily hormones. The study of the androgenic gland in decapodslags behind that in the isopods, and a decapod androgenic hormonehas yet to be identified. In this review, five decapod speciesare described as models, in which the androgenic gland exertsmorphological, anatomical, physiological and behavioral effects.These models could serve as the basis of possible bioassaysfor the study of the structure and mode of action of the androgenichormone in decapod crustaceans.