Formation of mature egg envelope subunit proteins from their precursors (choriogenins) in the fish, Oryzias latipes: loss of partial C-terminal sequences of the choriogenins

The inner layer of egg envelope of the medaka, Oryzias latipes, comprises two major groups of glycoprotein subunits, ZI-1,2 and ZI-3. Their precursor proteins, choriogenin H (Chg H) and choriogenin L (Chg L), respectively, are synthesized in spawning female liver. In the present study, the primary structures of the precursors and the corresponding mature subunits were compared by peptide mapping and amino acid sequencing to find what difference in their molecular structures is relevant to the assembly of the soluble precursors into the insoluble inner layer. The primary structures of the solubilized subunits were mostly identical to those of the respective precursors, but they lacked C-terminal partial sequences that their precursors possessed, namely, ZI-1,2 subunit was shorter than Chg H by 34 amino acid residues and ZI-3 was shorter than Chg L by 27 residues. In addition, a consensus amino acid sequence, Arg-Lys-X-Arg, was found at the putative cleavage sites in the C-terminal region of the precursors. It is conjectured that the truncation of the precursor proteins is prerequisite for formation of mature chorion subunit proteins and their assembly into chorion.     

Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity

Two vitelline envelope precursors (choriogenin H: Chg H; choriogenin L: Chg L) and an egg yolk precursor (vitellogenin B: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be approximately 215 kDa and approximately 69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to approximately 51 kDa and approximately 44 kDa, respectively. The mass of intact VgB was approximately 530 kDa and resolved into a polypeptide of approximately 185 kDa in SDS-PAGE. Specific antisera were raised against each purified protein and specific immunoassays were developed. When Chg H, Chg L and VgB were induced in the serum of immature mullet by injection with various doses of estradiol-17beta (E(2)), VgB exhibited the most sensitive response exhibiting high variation in its induced levels. The variation in induced levels of Chg H and L was relatively minimal although induction required higher doses of E(2) than with VgB. Serum samples obtained from immature mullet populations collected from their natural habitat exhibited similar profiles in the levels of these proteins. The present study suggests that the utilization of multiple biomarkers holds great importance for the reliable and accurate evaluation of estrogenic activity in aquatic environments.     

Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou

Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.     

A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes

A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the [125I]SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layer of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.     

Spawning Female-Specific Egg Envelope Glycoprotein-Like Substances in Oryzias latipes

In addition to the spawning female-specific (SF) substance (3, 6), a group of new higher molecular weight proteins cross-reacting with anti-egg envelope (chorion) glycoprotein (F1) antibody (original antibody) were found in the liver, blood plasma and the ovary of spawning female fish and in the ascites of the estrogenized fish of Oryzias latipes . Exploiting the antibodies specific for the SF substance and the new proteins, which were made of the original antibody by absorbing with the new proteins or the SF substance, the new proteins were found to behave very similarly to the SF substance concerning their localization in the inner layer of the oocyte envelope, intrahepatic formation in response to estrogen etc. They include the protein bands corresponding to Zl-1 and -2, two major constituent glycoproteins of the oocyte envelope, while the SF substance corresponds to ZI-3, the third major constituent of the envelope. Thus the three major constituent proteins of the inner layer of oocyte envelope are probably formed in the liver under the influence of estrogen in this fish.     

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved.     

Establishment of ELISA for murrel vitellogenin and choriogenin, as biomarkers of potential endocrine disruption

Vitellogenin (Vg) and choriogenin (Chg) are sensitive biomarkers for testing endocrine disruption in fish. Therefore, we have developed immunoassays for Vg and Chg in the Indian freshwater murrel, Channa punctatus. Vg is a known precursor of egg-yolk proteins, whereas Chg contributes to the formation of egg-envelope. Vg and Chg were induced in male murrel by administration of estradiol-17beta. Chg had an apparent native molecular mass of 180 kDa. It consisted of a single peptide with a molecular mass of 110 kDa, whereas native Vg protein (530 kDa) contained 175 kDa peptide. Highly specific polyclonal antibodies against purified plasma proteins, Vg and Chg, were employed for developing competitive enzyme linked immunosorbent assays (ELISAs). The sensitivity of Vg assay was 3.9 ng/mL (working range 15-500 ng/mL) and of Chg assay was 1.56 ng/mL (working range 6-200 ng/mL). The inter- and intra-assay variations were well within acceptable limits. The two antisera did not cross-react with male plasma proteins. Antiserum to Vg did not cross-react with Chg. Similarly, antiserum to Chg showed no correlation with Vg. Further, immunofluorescence and Western blotting confirmed the specificity of Vg and Chg antisera.     

Expression and localization of transcripts of MT5-MMP and its related MMP in the ovary of the medaka fish Oryzias latipes

cDNA clones of MT5-matrix metalloproteinase (MT5-MMP) and a related protein (designated MT5-MMP-del) were isolated by screening the cDNA library and by 3'-rapid amplification of cDNA ends using an ovary RNA of the medaka fish Oryzias latipes. The MT5-MMP clone encodes a protein of 546 amino acids while the MT5-MMP-del clone encodes a protein of 431 amino acids. Compared with mammalian counterparts, the fish MT5-MMP and MT5-MMP-del both lack the signal peptide and a part of the prodomain. The fish MT5-MMP and MT5-MMP-del were different in that the latter did not have the stem/transmembrane/cytoplasmic domain. The two fish MMPs were expressed in the ovary, testis, brain, and intestine. In the ovary, MT5-MMP mRNA was expressed in the oocytes of small growing follicles. In contrast, MT5-MMP-del mRNA was found in the stromal interstitial cells. These results strongly suggest that a MT5-MMP/gelatinase A cascade may possibly operate in the process of spawning and/or other events associated with ovulated oocytes or fertilized eggs of the medaka fish.     

Genomic structure and expression of the soluble guanylyl cyclase alpha2 subunit gene in the medaka fish Oryzias latipes

A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).     

Purification of serum precursor proteins to vitelline envelope (choriogenins) in masu salmon,Oncorhynchus masou

Three vitelline envelope-related proteins (VERPs), very-high-molecular-weight VERP (vhVERP), high-molecular-weight VERP (hVERP) and low-molecular-weight VERP (lVERP) were purified from female masu salmon serum. The apparent molecular weights of vhVERP, hVERP and lVERP, in their native state, were 520, 88 and 54 kDa, respectively, by gel-filtration chromatography. Very-high-molecular-weight VERP comprises two subunits, corresponding to 175 and 126 kDa. On SDS-PAGE, hVERP and lVERP migrate at 53 and 47 kDa, respectively. Amino acid analysis of vhVERP and hVERP showed that they share a high content of glutamic acid and proline. By contrast, lVERP is rich in glutamic acid and asparatic acid. These features are in good agreement with the amino acid composition of the vitelline envelope. Immuno-biochemical analysis suggested that vhVERP is derived from hVERP by polymerization and/or aggregation. Antibodies against hVERP and lVERP specifically immunostained the vitelline envelope and liver of female masu salmon. In addition, both hVERP and lVERP were induced in the serum of estrogen-treated male fish. Taken together, it is suggested that hVERP and lVERP are homologous molecules with choriogenin H and choriogenin L in medaka, respectively. These results indicate that hVERP and lVERP are precursor proteins to the vitelline envelope (choriogenins) in masu salmon.     

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low ( approximately 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61+/-0.08, 0.98+/-0.18 and 10.93+/-3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

Identification and characterization of a unique Xenopus laevis egg envelope component,ZPD

We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.     

Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor

A Drosophila cDNA clone was obtained using the human insulin receptor cDNA sequence as a probe. The 3586 bp nucleotide sequence predicted a single polypeptide of 1095 amino acid residues which showed considerable homology (35.2%) with the human insulin receptor precursor. Although the cDNA was incomplete at its 5'-terminal region, it encodes a transmembrane glycoprotein as a single precursor of a two subunit molecule having a structural architecture similar to that of the human insulin receptor precursor. The presumptive beta subunit carries a well conserved Tyr kinase domain which showed 63.5% homology with that of human insulin receptor; however the protein of the alpha subunit is only weakly conserved (25%).     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein.

Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize bt1 locus encodes an amyloplast membrane metabolite translocator protein (Sullivan, T. D., Strelow, L. I., Illingworth, C. A., Phillips, R. L., and Nelson, O. E., Jr. (1991) Plant Cell 3, 1337-1348). The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite translocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.