Deduced primary structure of vitellogenin in the giant freshwater prawn, Macrobrachium rosenbergii, and yolk processing during ovarian maturation

A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

Vitellogenesis in both sexes of gonochoristic mud shrimp, Upogebia major (Crustacea): analyses of vitellogenin gene expression and vitellogenin processing

The mud shrimp, Upogebia major is a gonochoristic species with distinct sexual dimorphism; however, the male has the “ovarian part of testis” in the gonad and mature-looking eggs appear in a similar reproductive cycle to the female. Vitellogenesis of U. major was investigated focusing on the characterization of vitellogenin (Vg) gene expression and Vg processing. Vg cDNA cloned by PCR-based methods was 7799 bp-long, encoding 2568 amino acids in a single open reading frame. The deduced amino acid sequence shared common characteristics conserved in other shrimp Vgs. The Vg gene was expressed in the hepatopancreas of females and males, the ovary, and the ovarian part of testis. Vitellins (Vns) were detected in the gonads of both females and males as three prominent polypeptides with apparent molecular masses of 82 kDa, 100 kDa, and 115 kDa. N-terminal amino acid sequences determined for the three polypeptides were present in the deduced amino acid sequence, demonstrating that they derived from a single long Vg polypeptide. Immunoblot analysis using polyclonal antibodies raised against two Vns (82 kDa and 100 kDa) confirmed Vg processing in the hepatopancreas, in the hemolymph and possibly in the oocytes, similarly in both sexes.     

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Vitellogenin of the cockroach,Leucophaea maderae: nucleotide sequence,structure and analysis of processing in the fat body and oocytes

A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced. The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide 'subunits' following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights = 112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A)+ RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.     

Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

Evidence for two vitellogenin-related genes in Leucophaea maderae: the protein primary structure and its processing

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.     

Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.     

Vitellogenin of the cicada Graptopsaltria nigrofuscata (Homoptera): analysis of its primary structure

We cloned and sequenced the cDNA of vitellogenin (Vg) from the cicada Graptopsaltria nigrofuscata (Homoptera). The deduced amino acid sequence of 1987 residues (including 16 residues for a putative signal peptide) was obtained. The pro-Vg was cleaved into two subunits between residues 379 and 380 following a consensus RXXR cleavage site sequence, secreted as S-Vg (apparent molecular weight 43 kDa) and L-Vg (200 kDa), sequestered, and stored in the egg as two vitellins (Vns), S-Vn and L-Vn, with similar respective molecular weights. There was a single long serine-rich stretch closely following the cleavage site. The entire amino acid sequences of the Vgs from the eight insects so far reported could be aligned confidently. The presence of subdomains I-V (areas of relatively high amino acid conservation) and of 10 cysteines at conserved locations at the C-terminus, noted previously among insect Vgs, were confirmed. Antisera raised against G. nigrofuscata S- and L-Vn cross-reacted with the S- and L-Vg/Vn, respectively, of all three other cicada species examined. Another major egg protein (170 kDa) unrelated to Vg/Vn, was also detected in all species examined.     

Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products

The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.     

Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses

A cDNA expression library constructed from poly (A)(+) RNA prepared from vitellogenic female fat body cells of the American cockroach, Periplaneta americana (Dictyoptera) was screened using a polyclonal antiserum against the 100-kD polypeptide(s) from the egg extract. A partial Vg cDNA clone was obtained and sequenced. The 5' end portion of the cDNA was then obtained by the RACE method, cloned, and sequenced. The combined complete Vg cDNA was 5,854 bp long and contained a single ORF encoding 1,896 amino acids. The entire deduced amino acid sequence was aligned confidently with those of the known insect Vgs. A GL/ICG motif, a number of cysteines at conserved locations following this motif, and a DGXR motif upstream of the GL/ICG motif were present near the C-terminal. The chemically determined N-terminal amino acid sequence of the 170-kD polypeptide from the egg extract completely matched the deduced sequence starting from just after one of the consensus (RXXR) cleavage sites, indicating the occurrence of post-translational cleavage in the fat body cells. The Vg gene begins to be expressed in the 2-day-old adult female fat body cells but is never expressed in ovaries or in male fat body cells. Hemolymph Vg was first detected by immunoblotting in 4-day-old adult females, 2 days after the beginning of gene expression. Western blot analysis of major yolk polypeptides in nine cockroach species belonging to the two superfamilies, Blattoidea and Blaberoidea, using the antisera against P. americana major yolk polypeptides showed that the similarities in Vn antigenicity are basically limited to within a superfamily.     

ANALYSIS OF THE Vitellogenin GENE OF RICE MOTH,Corcyra cephalonica STAINTON

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721‐bp long and contained 5five introns and six exons that together formed a 5,382‐bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16‐amino‐acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C‐terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed thatCceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, theCceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, theCceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection ofCceVg double‐stranded RNA into early‐emergent females caused severely abnormal ovaries.     

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.     

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.     

Two ferritin subunits from disk abalone (Haliotis discus discus): cloning, characterization and expression analysis

Ferritin plays a key role in cellular iron metabolism, which includes iron storage and detoxification. From disk abalone, Haliotis discus discus, the cDNA that encodes the two ferritin subunits abalone ferritin subunit 1 (Abf1) and abalone ferritin subunit 2 (Abf2) were cloned. The complete cDNA coding sequences for Abf1 and Abf2 contained 621 and 549 bp, encoding for 207 and 183 amino acid residues, respectively. The H. discus discus Abf2 subunit contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. Abf2 mRNA contains a 27 bp iron-responsive element (IRE) in the 5'UTR position. This IRE exhibited 96% similarity with pearl and Pacific oyster and 67% similarity with human H type IREs. However, the Abf1 subunit had neither ferroxidase center residues nor the IRE motif sequence; instead, it contained iron-binding region signature 2 (IBRS) residues. Recombinant Abf1 and Abf2 proteins were purified and the respective sizes were about 24 and 21 kDa. Abf1 and Abf2 exhibited iron-chelating activity 44.2% and 22.0%, respectively, at protein concentration of 6 microg/ml. Analysis of tissue-specific expression by RT-PCR revealed that Abf1 and Abf2 ferritin mRNAs were expressed in various abalone tissues, such as gill, mantle, gonad, foot and digestive tract in a wide distribution profile, but Abf2 expression was more prominent than Abf1.