Transcriptome analysis of female reproductive tissues of Anastrepha obliqua and molecular evolution of eggshell proteins in the fraterculus group.

The investigation of cDNA libraries has been an important tool for the identification of new genes in nonmodel species such as the fruit flies from the Anastrepha fraterculus group. In the present study, we constructed a cDNA library from the female reproductive tissues of Anastrepha obliqua aiming to identify genes with high evolutionary rates. We sequenced 2304 clones obtained from the female reproductive tissues of A. obliqua flies. The expressed sequence tags generated a total of 816 unigenes which were classified into different protein classes. Among these,we identified chorionic and vitelline protein genes as being among the most highly expressed. We used unigene sequences to amplify a set of chorionic and vitelline genes, involved in the formation of the eggshell,in species of the fraterculus group. Four chorionic genes and two vitelline genes showed evidence of positive selection along the Anastrepha and/or Tephritidae lineage. The signal of selection detected for Vm26Aa was possibly generated by a gene duplication event. The rapid evolutionary rates indicate that these genes could serve as important markers in population and evolutionary studies, not only for species of this group, but possibly also for other Diptera.

Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri.

BACKGROUND: Zona pellucida domain-containing proteins (ZP proteins) have been identified as the principle constituents of the egg coat (EC) of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP) structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata. RESULTS: Five ZP proteins (BbZP1-5) were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA) domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates showed that BbZP1-5 form a monophyletic group and share no significant sequence similarities with the ZP proteins of urochordates and the ZP3 subtype of jawed vertebrates. By contrast, small regions of homology were identifiable between the BbZP and ZP proteins of the non-jawed vertebrate, the sea lamprey Petromyzon marinus. The lamprey ZP proteins were highly similar to the ZP1 and ZP2 subtypes of the jawed vertebrates, which suggests that the ZP proteins of basal chordates most likely shared a recent common ancestor with vertebrate ZP1/2 subtypes and lamprey ZP proteins. CONCLUSIONS: The results document the spectra of zona pellucida domain-containing proteins of the egg coat of basal chordates. Particularly, the study provides solid evidence for an invertebrate origin of vertebrate ZP proteins and indicates that there are diverse domain architectures in ZP proteins of various metazoan groups.

The chicken or the egg: PHEX, FGF23 and SIBLINGs unscrambled.

The eggshell is an ancient innovation that helped the vertebrates' transition from the oceans and gain dominion over the land. Coincident with this conquest, several new eggshell and noncollagenous bone-matrix proteins (NCPs) emerged. The protein ovocleidin-116 is one of these proteins with an ancestry stretching back to the Triassic. Ovocleidin-116 is an avian homolog of Matrix Extracellular Phosphoglycoprotein (MEPE) and belongs to a group of proteins called Small Integrin-Binding Ligand Interacting Glycoproteins (SIBLINGs). The genes for these NCPs are all clustered on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of the SIBLING proteins is an Acidic Serine Aspartate-Rich MEPE (ASARM)-associated motif. The ASARM motif and the released ASARM peptide play roles in mineralization, bone turnover, mechanotransduction, phosphate regulation and energy metabolism. ASARM peptides and motifs are physiological substrates for phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), a Zn metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets. PHEX interacts with another ASARM motif containing SIBLING protein, Dentin Matrix Protein-1 (DMP1). DMP1 mutations cause bone-renal defects that are identical with the defects caused by loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both X-linked hypophosphatemic rickets and ARHR, increased fibroblast growth factor 23 (FGF23) expression occurs, and activating mutations in FGF23 cause autosomal dominant hypophosphatemic rickets (ADHR). ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. This review will discuss the evidence for a new integrative pathway involved in bone formation, bone-renal mineralization, renal phosphate homeostasis and energy metabolism in disease and health.

SUEL-related lectins, a lectin family widely distributed throughout organisms.

Glycan-binding proteins are categorized into two groups, lectins and sulfated glycosaminoglycan-binding proteins. SUEL-related lectins are members of a superfamily of proteins containing a carbohydrate-recognition domain (CRD), which is structurally similar to sea urchin egg lectin (SUEL). Here I review the structure and function of this family of proteins.

Purification and classification of three lipovitellin subtypes in the marbled sole (Pleuronectes yokohamae).

An immunologlcal analysis using subtype-specific antisera of the major yolk protein lipovltellin (Lv) of the grey mullet (Mugil cephalus) confirmed the presence of the three corresponding Lv subtypes (LvA, LvB, and LvC) in vitellogenic ovaries of the marbled sole (Pleuronectes yokohamae). These three Lv subtypes were purified from sole ovaries by using various combinations of anion exchange, hydroxylapatite, immunoadsorbent, and gel-filtration chromatography. Purified LvA, LvB, and LvC had an apparent native mass of approximately 482, approximately 380, and approximately 372 kDa, respectively, estimated by gel filtration. Analysis of their tertiary structures by SDS-PAGE indicated that LvA, LvB, and LvC were typical of teleost Lvs in having a heavy (H) chain (approximately 105, approximately 102, and approximately 107 kDa, respectively) and a light (L) chain (approximately 22, approximately 19.5, and approximately 25 kDa, respectively). The N-termlnal amino acid (AA) sequences were obtained for the LvA H chain, the LvB H and L chains, and the LvC L chain and compared to the deduced AA sequences of their precursors, vitellogenins (Vgs), in several species. This comparison of LvA, LvB, and LvC with various teleost VgA, VgB, and VgC sequences, respectively, revealed high identities (60-100%). The purified Lv subtypes were subjected to double immunodiffusion using an antiserum against an unclassified Lv of the sole ( Hashimoto et al., 1998 ); only the LvB subtype exhibited immunoreactivity with this antiserum. This result indicates that the previously developed immunoassay using this anti-Lv for the detection of sole Vg is effectively a VgB-specific assay.

The functions of the multiproduct and rapidly evolving dec-1 eggshell gene are conserved between evolutionarily distant species of Drosophila.

The Drosophila dec-1 gene encodes multiple proteins that are required for female fertility and proper eggshell morphogenesis. Genetic and immunolocalization data suggest that the different DEC-1 proteins are functionally distinct. To identify regions within the proteins with potential biological significance, we cloned and sequenced the D. yakuba and D. virilis dec-1 homologs. Interspecies comparisons of the predicted translation products revealed rapidly evolving sequences punctuated by blocks of conserved amino acids. Despite extensive amino acid variability, the proteins produced by the different dec-1 homologs were functionally interchangeable. The introduction of transgenes containing either the D. yakuba or the D. virilis dec-1 open reading frames into a D. melanogaster DEC-1 protein null mutant was sufficient to restore female fertility and wild-type eggshell morphology. Normal expression and extracellular processing of the DEC-1 proteins was correlated with the phenotypic rescue. The nature of the conserved features highlighted by the evolutionary comparison and the molecular resemblance of some of these features to those found in other extracellular proteins suggests functional correlates for some of the multiple DEC-1 derivatives.

[Problem of the rational classification of dietary proteins]. / K voprosu o ratsional'noi klassifikatsii pishchevykh belkov.

A classification of food proteins has been proposed based on two new qualitative parameters: potential biological value (BVp) and compensation coefficient (C). BVp determines the balance degree and agreement with the body requirement of food protein amino acids. The C coefficient estimates the value of the food protein aminogram improvement at the expense of the endogenous essential amino acid reserve. According to these parameters food proteins can be divided into 4 classes. Class I includes proper alimentary specific food proteins (milk and egg proteins) possessing mean values of BVp and high C coefficients. Class II proteins are characterized by rather high BVp values, i.e. by a good balance of essential amino acids and C coefficient reaching 0, they are represented by animal proteins (those of meat and fish), and vegetable proteins (those of soybean, rape and cotton). Class III contains proteins of food grains characterized by low BVp values and C coefficients. Class IV contains proteins with zero values of BVp, containing no essential amino acids (for example, gelatin, hemoglobin, zein), but showing high C coefficients. In such cases the nature of compensation differs from that of class I proteins, it is paradoxic and temporary.

Expression of zona pellucida B proteins in juvenile rare minnow (Gobiocypris rarus) exposed to 17α-ethinylestradiol, 4-nonylphenol and bisphenol A.

Zona pellucida (ZP) containing proteins are glycoproteins in teleost chorion and are encoded by several gene subfamilies, mainly including ZPA, ZPB, ZPC and ZPX genes. In teleost species, ZP genes are expressed either in liver under regulation of estrogen or in ovary. In the present study, five ZP gene isoforms were isolated and characterized in Gobiocypris rarus. The putative amino acid sequences of these ZP gene isoforms contain the typical trefoil motif and a ZP domain. These five G. rarus ZP gene isoforms were named as grZPB.1, grZPB.2, grZPB.3, grZPB.4 and grZPB.5. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis indicated that all these ZP mRNA isoforms were exclusively expressed in ovary. G. rarus juveniles at the age of 21 days postfertilization were exposed to 17α-ethinylestradiol (EE2; 0.01, 0.1 and 1 nM), 4-nonylphenol (4-NP; 10, 100 and 1000 nM) or bisphenol A (BPA; 0.1, 1 and 10nM) for 3 days. mRNA expressions of ZPB isoforms following the exposure to xenoestrogen were detected by RT-qPCR. Data were analyzed by the 2(-△△Cq) method. The results indicate that induction by 0.1-1nM EE2 on mRNA expression of the grZPB isoforms is weaker than for vitellogenin. 4-NP exposures at three concentrations had differential effects on the grZPBs. BPA at three concentrations weakly induced mRNA expression of the grZPB isoforms.

Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica.

A fish egg envelope is composed of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among vertebrate species. Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte. We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala. Five major components of the egg envelope were purified and their partial amino acid sequences were determined by sequencing. cDNA cloning revealed that the eel egg envelope was composed of four ZPC homologues and one ZPB homologue. Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins. This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution. Northern blotting and RT-PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver. Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver. Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway to Euteleostei.

The primary structure of a novel riboflavin-binding protein of emu (Dromaius novaehollandiae).

Emu riboflavin-binding protein (RBP) was purified from egg white and yolk, and its N-terminal amino acid sequence was determined. The molecular mass of emu RBP was estimated at approximately 48 and 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., 10 kDa larger than chicken RBP. The molecular mass of deglycosylated RBPs indicated that the content of oligosaccharide chain in emu RBP was approximately 3 times greater than that in chicken RBP. The gene encoding the RBP precursor was cloned from emu oviduct cDNA by PCR and found also in the liver and ovary cDNAs as well as oviduct cDNA. The complete cDNA consisted of an open reading frame of 714 bp encoding a protein of 238 amino acids. The amino acid sequence deduced from the cDNA sequence revealed that many essential structural features were conserved in emu RBP including 18 cysteine residues, 2 N-glycosylation sites, a clustered phosphorylation region, and riboflavin-binding sites. Two additional potential N-glycosylation sites were found in the amino acid sequences of RBPs from the emu and other sources such as the turtle and frog, which might in part account for the greater content of oligosaccharide chain of emu RBP as compared to chicken RBP.

Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4.

The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nucleotides encoding a polypeptide of 543 amino acid residues. The deduced amino acid sequence of ZP4 revealed high overall homology with rat (82%) and human (78%). Subsequent mass spectrometric analysis of the hamster ZP allowed identification of peptides from all four glycoproteins. The data presented in this study provide evidence, for the first time, that the hamster ZP matrix is composed of four glycoproteins.

Precursor structure of egg proteins in the coral Galaxea fascicularis.

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.

Drosophila Cornichon acts as cargo receptor for ER export of the TGFalpha-like growth factor Gurken.

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFalpha growth factor Gurken (Grk) to the oocyte surface. Here, we show that Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and propose that Cni acts as a cargo receptor recruiting Grk into COPII vesicles. Consequently, in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic processing of Grk still occurs in cni mutant ovaries, demonstrating that release of the active growth factor from its transmembrane precursor occurs earlier during secretory transport than described for the other Drosophila TGFalpha homologues. Massive overexpression of Grk in a cni mutant background can overcome the requirement of Grk signalling for cni activity, confirming that cni is not essential for the production of the functional Grk ligand. However, the rescued egg chambers lack dorsoventral polarity. This demonstrates that the generation of temporally and spatially precisely coordinated Grk signals cannot be achieved by bulk flow secretion, but instead has to rely on fast and efficient ER export through cargo receptor-mediated recruitment of Grk into the secretory pathway.

Cracking the egg: increased complexity in the zona pellucida.

A functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.

Evolution of the silk moth chorion gene superfamily: gene families CA and CB.

The nucleotide sequences of two developmentally early chorion cDNA clones from Bombyx mori define two distinct proline-rich chorion protein families, which we name CA and CB to indicate their homologies to the previously defined chorion protein families A and B, as well as the developmentally late and cysteine-rich HcA and HcB chorion families. Thus, the chorion gene superfamily has two symmetrical branches, each consisting of three families: the alpha branch (A, CA, HcA families) and the beta branch (B, CB, HcB families). The evolution of the superfamily is discussed.

Determination of amino acid sequence and site of mRNA expression of four vitellins in the giant freshwater prawn, Macrobrachium rosenbergii.

Four major yolk proteins, designated as vitellins (Vns) Macr-VnA, B, C, and D, were extracted from mature ovaries of Macrobrachium rosenbergii. These were purified to homogeneity by reversed-phase high performance liquid chromatography (HPLC) employing a unique separation system based on the hydrophobic properties of the Vn molecule. Using standard techniques of protein sequencing, more than 33 N-terminal and 57 internal amino acid residues were determined for each of the four Vns. The cDNA fragments encoding the four Vns were amplified by PCR using degenerate oligonucleotide primers derived from the N-terminal and internal amino acid sequences. These cDNA fragments were cloned, sequenced, and used as probes to examine the transcription of mRNAs encoding the four Vns. Significant accumulations of these mRNAs were observed in female hepatopancreas only, while mRNA expression was not detected in male hepatopancreas or any other female tissue including ovary, subepidermal adipose tissue, gill, and muscle. This is the first occasion in Crustacea in which multiple Vns were demonstrated to be synthesized simultaneously in a single tissue.

cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae.

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.