Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri
© Xu et al.; licensee BioMed Central Ltd. 2012
Received: 8 October 2012
Accepted: 29 November 2012
Published: 8 December 2012
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.
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.
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.
KeywordsAmphioxus Zona pellucida protein Proteomics Molecular evolution Sperm-egg interaction
Almost all metazoan eggs are surrounded by a proteinaceous matrix that is referred to as the zona pellucida (ZP) in mammals and the vitelline coat (VC) in non-mammals. ZP/VC proteins play important roles in fertilization and provide a protective barrier for oviparous animals, such as the amphioxus, fishes and amphibians. The family of ZP proteins is characterized by a conserved protein-protein interaction module, the ZP module [1–3]. The ZP module can be divided into two related domains, ZP-N and ZP-C, and the latter domain associates with the external hydrophobic patch (EHP) . The three-dimensional structure of the mammalian ZP3 shows that the EHP lies at the interface between the ZP-N and ZP-C domains, which are connected by a long loop that carries a conserved O-glycan important for sperm binding [5–7]. The dissociation of EHP from the ZP-C domain allows the ZP proteins to polymerize on the surface of oocytes, thus forming the extracellular coat [5, 6, 8].
ZP proteins have been characterized from diverse metazoan taxa, including many major groups of jawed vertebrates, urochordates and molluscs. In mammals, the ZP proteins are a family of 3–4 genes divided into 3 subtypes . However, the lower vertebrates, including fish [10, 11], birds  and amphibians [13–17], typically contain a greater number of ZP genes and subtypes .
In this study, we characterized the protein composition from the egg coat of a cephalochordate species, B. belcheri, by mass spectrometry, from which we identified multiple ZP proteins. We further examined the tissue distribution of the transcripts and mature proteins and observed that they are predominantly expressed in the developing eggs and localized in the cortical granules and extracellular spaces surrounding the eggs. We further identified homologous ZP protein genes from a Cyclostome, Petromyzon marinus, to trace the evolutionary relationship of the amphioxus ZP proteins with those of vertebrates. A sequence comparison of the ZP domains among the ZP proteins of gnathostome and cyclostome vertebrates, urochordates and cephalochordates showed that cephalochordate egg coat ZP proteins shared higher sequence similarities with vertebrates than urochordates and reliably suggested a distant homology between the cephalochordate and vertebrate ZPs. Therefore, the chordate egg coat ZP proteins might have a common origin deeply rooted in the lower invertebrates.
SDS-PAGE analyses of proteins from unfertilized and fertilized B. belcheriegg coats
LC-MS/MS identification of zona pellucida domain-containing proteins from the egg coats
Zona pellucida domain-containing proteins identified from B.belcheri unfertilized and fertilized egg coats by LC-MS/MS
To reduce the contamination from the cytoplasmic proteins and to enrich the egg coat protein components for the mass spectrometry analysis, we purified the ECs from the fertilized eggs, which are well separated from the cytoplasmic mass after egg coat expansion. These EC extracts were separated by PAGE and sliced into 5 pieces for LC-MS/MS analysis (Figure 2D). Consistent with what was observed from the unfertilized egg ECs, we identified only the same 5 ZP domain containing proteins from the fertilized egg ECs (Table 1, the right panel), which suggests that the 5 BbZPs compose the complete set of ZP components present in B. belcheri ECs. In addition, similar to situations observed in the unfertilized egg samples in which variants of BbZP1 and BbZP3 appeared in multiple gel slices, three BbZPs (BbZP1, BbZP3 and BbZP4) were detected in all 5 gel slices subjected to mass spectrometry in the fertilized egg samples (Table 1, right panel), which suggests the presence of molecular variants with different molecular weights from these gene products.
The spectral counts observed in the mass spectrometry analysis have been suggested to approximately quantify the abundance of each protein in a sample [29–31]. Among the five identified ZP proteins, BbZP1, BbZP3 and BbZP4 showed much higher spectral counts than BbZP2 and BbZP5, which suggests that these three proteins are the major ZP protein types constituting the fertilized egg coat and unfertilized eggs (Table 1). In addition to the zona pellucida domain-containing proteins, a number of proteins were detected in the fertilized egg ECs with spectral counts greater than 10 but fewer than those of BbZPs (Additional file 1: Table S1). These proteins include a multiple EGF-like domain protein, three vitellogenins, one zonadhesin-like protein, one melanotransferrin-like protein, a matrilin and an apolipoprotein B-like protein. A recent study showed that in the ascidian Halocynthia roretzi, vitellogenin is a component of the vitelline coat and participates in fertilization as the egg-coat binding partner of sperm proteases . In addition, an apolipoprotein B-like protein has been demonstrated to reside both on the VC and in the egg cytoplasm of the ascidian C. intestinalis. Therefore, a few other proteins are commonly present in the ECs of cephalochordates and urochordates in addition to multiple ZP proteins.
Characterization of the B. belcheriZP genes
We performed RT-PCR and 5’ and 3’ RACE using various primer sets and then sequenced the RT-PCR products to obtain the full-length coding sequences of the five ZP domain-containing proteins. The full-length cDNA of each ZP gene was obtained by piecing together the overlapping fragments. The translated proteins from the full-length BbZP1, BbZP2, BbZP3, BbZP4 and BbZP5 transcripts are 927, 697, 687, 891 and 673 amino acids, respectively. The sequence alignment of the 5 ZP proteins is shown in Additional file 2: Figure S1.
The above data revealed significant discrepancies between the calculated molecular weights of the predicted full-length BbZPs and the positions each protein migrates on the polyacrilamide gel (i.e., the gel slices in which the proteins were detected by mass spectrometry). Among the 5 BbZPs, the calculated MWs range between ~75 kDa (BbZP5) and ~100 kDa (BbZP1). However, for the unfertilized egg EC sample, protein segments of BbZP1 are abundantly detected in the gel slices that supposedly contained proteins larger than 100 kDa (slices 1–5 of Figure 2C), whereas peptides of BbZP3, BbZP4 and BbZP5 were detected in the gel slices (slices 8–11 of Figure 2C) supposedly containing only proteins with MWs smaller than 75 kDa (Table 1, Figure 2C). The discrepancy is more pronounced in the fertilized EC sample, in which protein fragments of three BbZPs, BbZP1, BbZP3 and BbZP4, are observed in the gel positions (slices 1 and 2 of Figure 2D) for proteins with MWs greater than 120 kDa, whereas the peptides from all 5 BbZPs were detected in gel slices that should have contained proteins with MWs less than 55 kDa (Table 1, Figure 2D). A western blot analysis of the fertilized egg EC extracts, using specific antibodies against the 5 BbZPs, further verified the high polymorphic nature of BbZP1, BbZP3 and BbZP4 (Additional file 4: Figure S3).
Egg coat ZP proteins are well known to be highly glycosylated [5, 10, 33], which may contribute to the higher than expected MWs in the SDS-PAGE analysis. To determine how potential glycosylation might have affected the BbZPs, we treated the fertilized egg EC extracts with the enzyme PNGase F, which was specific to cleave the N-linked glycosylation moieties off of the proteins. The overall gel migration pattern of the treated sample remained the same, except that the 55 kDa band in the untreated sample narrowly separated into two 55 kDa bands, the faint 37 kDa band in the untreated sample disappeared, and a smaller (~36 kDa) band appeared (Additional file 5: Figure S4). These results indicate that glycosylation, at least N-glycosylation, is not the major factor in the observed aberrant migration patterns of the BbZPs. It is possible that the incomplete disassociation of the BbZP polymers may have resulted in the higher than calculated MWs observed in the polyacrylamide gels.
We checked whether alternative splicing of the BbZP transcripts could occur to understand why some BbZPs appeared in the gels with smaller than calculated MWs. Within sets of specific primers of BbZP1 and BbZP4, we identified cDNA variants that were shorter than expected from the full-length cDNAs (Additional file 6: Figure S5), indicating the presence of alternative splicing. The current survey of the BbZP transcripts is not exhaustive, and more alternatively spliced forms of BbZPs will likely be identified if more complete RT-PCR experiments are performed. However, the smaller sized BbZP variants could be a result of proteolysis of the full-length BbZP precursors. For example, the vitelline coat ZP protein, HrVC70, is derived from HrVC120, which is a larger VC ZP protein in the urochordate H. roretzi[22, 23].
Tissue specific expression and cellular localization of the BbZPs
B. belcheri ZP proteins showed sequence homology with the non-jawed vertebrate, Petromyzon marinus
Phylogenetic analyses showed that ZP genes of the basal chordate form a distinct evolutionary clade and most likely share a recent common ancestor with the lamprey ZP proteins and ZP1/2 subtypes of the high vertebrates
Using comprehensive proteomic approaches, we identified 5 proteins containing a zona pellucida domain from B. belcheri egg coat extracts (Table 1). We further verified the physical location of these proteins on the egg coat by immunohistochemistry using antibodies raised to specifically target these ZP proteins (Figure 5B). A search for homologous genes in the published B. floridae genome revealed that all 5 of the B. belcheri genes have B. floridae orthologs and that each pair shared more than 80% protein sequence similarity. In addition, the genomic locations and relative orientations of the ZP orthologs from the two species are highly conserved (Additional file 8: Figure S7). In B. belcheri, a search of the newly sequenced genome showed that BbZP1 and BbZP3 form a synteny in scaffold 3, BbZP2 and BbZP5 co-localize in scaffold 88, and BbZP4 localizes in a separate scaffold (Scaffold 33). Notably, the genes in Scaffolds 3 and 33 were highly expressed, whereas the ones in scaffold 88 were not. Similarly, in the B. floridae genome, the orthologs of BbZP1 and BbZP3 (BfZP1 and BfZP3) are observed in scaffold Bf_V2_271, ~23 kb apart from one another; the orthologs of BbZP2 and BbZP5 (BfZP2 and BfZP5) are linked in tandem in scaffold Bf_V2_78 with ~3 kb between the two. A small difference occurs in the case of BbZP4. Whereas we identified only one BbZP4 gene in the B. belcheri genome, two orthologs (BfZP4-1 and BfZP4-2) were observed in B. floridae, arranged in a head-to-head orientation in scaffold Bf_V2_243 in the B. floridae genome, a scaffold that is distinct to BbZP1/3 and BbZP2/5. A genomic comparison showed that the ZP protein genes identified in this study are common to the genus Branchiostoma. The cephalochordates comprise approximately 35 species that are divided into three genera: Branchiostoma, Epigonichthys and Asymmetron. The distribution of these genes among other genera of Cephalochordata requires further study.
In recent years, ZP proteins have been characterized from urochordates, which are phylogenetically the closest relatives of vertebrates. Notably, comparisons of the MS results of B. belcheri with those from C. intestinalis reveal that the number of ZP proteins identified in this study is significantly less than those observed in C. intestinalis (5 vs 11), which suggests that the number of ZP protein genes in the basal chordate species might be fewer than those of the urochordates. In addition to the ZP proteins, there are some non-ZP proteins that might also be constituents of the egg coat, for example, vitellogenin and apolipoprotein B-like protein, which are also the known components of egg coats in urochordates [22, 32]. Whereas mammals use ZP proteins as the sole components to compose the zona pellucida matrix that surrounds the egg, the lower chordates appear more variable in selecting the egg coat composition.
ZP proteins have been observed to be the major constituents of egg coats in diverse metazoan groups (Figure 1); however the evolutionary relationship among ZP proteins is not obvious. The identification of ZP proteins as important components of the cephalochordate egg coats has filled a gap in the knowledge regarding chordate ZP proteins and has enabled us to make conjectures regarding the evolutionary processes of ZP proteins in chordates. Both the sequence comparison (Figure 6) and ZP domain tree (Figure 7) indicated that cephalochordate ZP proteins are evolutionary homologues of the lamprey ZP proteins and the ZP1, ZP2 and ZPAX subtypes of the jawed vertebrates, which suggests a common ancestor for the two ZP clades. Therefore, vertebrate ZPs appeared to have an invertebrate origin (i.e., at least began at the base of chordate evolution) rather than an independent recruitment of ZP domain containing proteins. However, the urochordate ZP proteins appeared to be closer to the ZP3/ZPC subtypes of vertebrates (Figure 7). The phylogenetic tree of ZP proteins from the three chordate subphyla indicated that the vertebrate ZP subtypes had two recently separated common ancestors. In addition, except for the ZP modules, which are the common structural domains of all metazoan ZP types, the other domain components of the ZP proteins from cephalochordates, urochordates and vertebrates are different. Von Wallebrand and repetitive EGF domains are found in the egg coat ZP proteins from cephalochordates and urochordates, respectively, which suggests different domain structures may be involved in gamete recognition in the two groups.
The high resolution structures of the mouse ZP3  and ZP-N domains  provided an understanding of the structural basis of sperm recognition in mammals. Furthermore, the accumulating evidence suggests that the presence of repeated ZP-N domains in the ZP proteins, in addition to the universal ZP-module, may well be associated with the sperm-binding activity . A recent threading analysis of the VERL repeats in abalone ZP proteins suggested that, similar to their ZP2 counterparts, the VERL repeats most likely adopt a ZP-N fold, as shown by the complete conservation of four cysteine residues within each repeat . The domain architecture of the 5 BbZP proteins (Figure 3) shows that they possess neither the EGF-like domain repeat nor the ZP-N repeats. Most of the lamprey ZP proteins identified in this study are not full-length because of the low coverage of the current available genome. The full-length lamprey ZP2 also lacks an apparent ZP-N domain or an EGF-like domain; therefore, whether or how the BbZP proteins and lamprey ZP proteins function in sperm recognition during fertilization remains an open question that warrants further investigations.
By comprehensive proteomic analysis, followed by in situ hybridization and immunohistochemical analyses, we identified five egg coat ZP proteins from the cephalochordate B. belcheri. We also identified four ZP proteins from the jawless vertebrate, the sea lamprey, which are highly similar to vertebrate ZP subtypes. Molecular phylogenetic analyses showed that the B. belcheri ZP proteins form a distinct evolutionary clade but are homologous to both the lamprey and vertebrate ZPs in protein sequences. The study traces the evolutionary history of vertebrate sperm-egg recognition molecules to the appearance of basal chordate animals in the metazoan phylogeny.
The amphioxus used in this study were captured from the Xiamen Tong’an coastal waters of the East China Sea during the spawning season (March-October) and transferred in sand mixed with seawater to Shanghai Ocean University for species identification. B. belcheri and B. japonicum were identified by their morphological traits. The animals were screened for their sex and stage of gonad development, and then stored individually at −80°C for further use. The experimental procedures are followed with the guidelines established by the Ethic Committee for Animal Usage in Research of Shanghai Ocean University where the animal procedures are carried out.
Egg coat separation
To obtain the egg coats (ECs) of unfertilized eggs, fully developed eggs were surgically separated from the gonad and then shaken in Ca2+/Mg2+-free artificial seawater until the eggs were fully separated. The eggs were then gently homogenized in 0.2×Ca2+/Mg2+-free artificial seawater (4 mM EPPS [1-Piperazinepropanesulfonic acid, 4-(2-hydroxyethyl)], pH 8.0, 92 mM NaCl, and 2 mM KCl) containing a protease inhibitor mixture (1 mM phenylmethylsulphonyl fluoride, 10 μg/ml leupeptin) with a Teflon homogenizer. The homogenate was filtered through a nylon mesh (22 um). The EC that remained on the mesh was washed by pipetting, using 0.2×Ca2+/Mg2+-free artificial seawater containing 0.005% Triton X-100, and further purified manually under a binocular microscope. To obtain the ECs of fertilized eggs, the elevated coats of 20 fertilized eggs were manually peeled and thoroughly rinsed with 0.2×Ca2+/Mg2+-free artificial seawater at least 5 times. Both the unfertilized and fertilized EC samples were extracted with Laemmli SDS-PAGE sample buffer containing 5% 2-mercaptoethanol and were denatured by boiling for 5 min prior to SDS-PAGE analyses.
The egg coat extracts from unfertilized and fertilized eggs, each with approximately 20 μg proteins, were separated using 15% SDS-PAGE and 1.5 mm-thick gels. After electrophoresis, the gels were stained with Coomassie Blue R250 (Sigma, USA). The gel of the unfertilized eggs was dissected into 11 slices and that of the fertilized eggs into 5 slices (Figure 2) for LC-MS/MS analyses.
Western blot analysis
The protein concentration of the egg extracts from the fertilized eggs was measured using a BCA protein assay kit (Pierce, Rockford, IL). The egg extracts were subjected to electrophoresis in 12% SDS-PAGE gel. The gels were transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) skimmed milk in TBS overnight at 4°C. The blocked membranes were incubated separately with a primary antibody, namely, polyclonal rabbit Anti-BbZP1, Anti-BbZP3 (diluted 1:3000) or mouse polyclonal antibodies against BBZP2, 4 and 5 (diluted 1:4000) in TBS containing 5% skimmed milk for 1 hr at room temperature. The incubated membranes were then washed with TBS-T 3 times for 15 minutes each. The membranes were incubated with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody (1:5000; Santa Cruz, CA) for 1 hr at room temperature and washed with TBS-T. The blots were visualized by using the Immobilon Western chemiluminescence HRP substrate system (Pierce, Rockford, IL) following exposure to medical X-Ray films (Fuji film, Tokyo, Japan).
Enzyme digestion, LC-MS/MS analysis and database searching
The dissected, protein-containing gel blocks were subjected to trypsin treatment (0.2 μg, trypsin in 25 mm NH4HCO3 buffer at 37°C overnight). The digested peptides were extracted by 50% acetonitrile and 5% formic acid at room temperature for 30 mins . The digested products from each gel band were then separated on a Paradigm MS4N Nano/Capillary HS MDLC (Michrom Bioresources, Inc. USA) using a 100 μm x 150 mm C18 reverse phase column. Liquid chromatography was conducted with a linear gradient of buffer A and 5–35% buffer B (50 min) followed by 35–90% buffer B (10 min) and 90% buffer B for 10 minutes at a flow rate of 500 nl/min. Buffer A comprised 0.1% formic acid in a 2% acetonitrile H2O solution, and buffer B was 0.1% formic acid in a 98% acetonitrile H2O solution. The separated peptides were then subjected to mass spectrometry analysis in a LTQ-MS (Thermol, USA) machine coupled with a Michrome Advanced nanospray apparatus (Microm Bioresources Inc.). The peak list files were generated using the Bioworks software (Applied Biosystems, USA) with the default parameters. The m/z peaks were searched against the predicted protein database (version 1.1) derived from the B. belcheri genome project (the draft genome of Branchiostoma belcheri: http://mosas.sysu.edu.cn/genome/) using the Sequest software. The parameters were established as follows: Xcorr ≥ 2 for two or three valent ions; Xcorr ≥ 1.5 for one valent ion; Deltacn ≥ 0.1; and at least two nonredundant peptides can be identified in a single protein. The false discovery rate was estimated as <1% using a reversed proteome database as a control. The mass spectrometry data was supplied in the additional file 9: Figure S8. The mass spectrometry data was deposited in the PRIDE database (http://www.ebi.ac.uk/pride/) under the reference No. 1-20121203-123403.
Characterization of the full-length transcripts of the zona pellucida domain-containing genes
The total RNA from stage IV B. belcheri ovaries was extracted using Trizol (Takara) following the manufacturer’s protocol. Two μg of the total RNA was reverse transcribed by SuperScript® III Reverse Transcriptase (Invitrogen, USA) in 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 5 mM MgCl and 5 mM DTT and zona pellucida domain-containing genes were amplified by polymerase chain reaction (PCR) using pairs of primers designed according to the predicted transcripts of the target genes. To obtain the UTR sequences, both 3’ and 5’ RACE were performed with the SMART RACE kit (TaKaRa Co, Japan) following the manufacturer’s instructions. The PCR products were cloned into the pGEM-easy TA cloning vector and sequenced; the overlapping clones were pieced together to gain the full-length cDNAs of the BbZP genes.
Animal section preparation
The fully developed gonads from adult B. belcheri animals were cut into small fragments (approximately 1 centimeter each) and fixed in 4% paraformaldehyde (PFA) and phosphate buffered saline (PBS, pH 7.4) for approximately 4 hr at room temperature. After dehydration in an ascending series of ethanol and clearing in xylene, the tissues were embedded in paraffin, sectioned transversely at 4–5 μm, and mounted on glass slides (RNase-free), which were precoated with polylysine. Because the sections were used for both in situ hybridization and immunohistochemistry, extra caution to avoid RNase contamination was taken.
One fragment for each gene was amplified and cloned into the pGEM-T vector (Promega). After verifying the clones by sequencing, the plasmids were purified and cut at one end using the appropriate restriction enzyme. The antisense riboprobe for each gene was synthesized using SP6 or T7 polymerase. The paraffin sections were used to examine their expression patterns as described by Yu and Holland .
Antibody generation and immunohistochemical staining
The divergent regions of BbZP2, 4, and 5 were expressed in bacteria using the pET-28a vector, and the His-tagged recombinant proteins were purified using a nickel bead column according to the instruction manual (Promega) and verified by SDS-PAGE electrophoresis. The purified recombinant proteins were injected into mice to produce polyclonal antibodies, and following the fourth injection (given at one week intervals), the mice were sacrificed for antisera. Anti-BbZP1, Anti-BbZP3 polyclonal antibodies were generated by immunizing rabbit with synthetic peptides. The specificity of the antibodies was verified by a western blot of the specific recombinant protein with bovine serum albumin as a control. Antibody batches with the best specificity were purified. The antibody raising and purification were conducted by Hua-An Biotechnology Inc. in Hangzhou, China. The purified antibodies were used for immunohistochemical staining. The paraffin sections were used to localize the expression regions of the tissues. Immunocytochemical staining was performed with an ABC (avidin-biotin peroxidase complex) kit (Maixin-Bio Co, China) as described by Bočina1 and Saraga-Babić .
The total RNA was extracted from B. belcheri gills, skin, muscles, livers, intestines, notochord, testes and ovaries and then reverse transcribed. Two pairs of primers for each ZP gene were designed and tested for the amplification efficacy and specificity by electrophoretic analysis of the PCR products. The primer pair with the best specificity was selected for further use in a Quantitative PCR (Q-PCR). Q-PCR was performed using the SYBR RT-PCR kit in a Bio-Rad CFX 96 (Bio-Rad) machine, and the results were analyzed by CFX Manager software. The PCR was performed using 45 cycles of 95°C for 15 seconds, 72°C for 30 seconds and 60°C for 30 seconds. The PCR reaction for each gene was performed in triplicate with the housekeeping gene beta-actin as the control.
The representative ZP genes from the urochordate Ciona intestinalis, the teleost fish Oryzias latipes, and the rodent Mus musculus were collected from NCBI GenBank entries. The teleost fish ZP genes were used as queries to search against the predicted lamprey cDNA databases (version Petromyzon_marinus_7.0), and transcripts with significant similarity (e-value ≤ 1e-20) were identified as ZP protein homologues. The ZP domain region of each ZP protein was delineated by searching against the SMART database . The ZP domain regions were aligned using t-coffee . A Bayesian inference of phylogeny was performed using MrBayes 3.2.1 (John P. Huelsenbeck and Fredrik Ronquist, MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755). The model selected by MrModeltest 2.3  according to AIC criterion was GTR+I+G. The tree was displayed using the Treeview software.
Epithelial growth factor
Von Willebrand factor type A.
The authors thank Dr. Yingchun Wang's group in the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences for performing part of the mass spectrometry analysis reported in this paper.
We thank Yong Liu for assistance with collecting the amphioxus specimens. We are also grateful of Mr. Bing Gu, currently in the College of Life Sciences, Zhejiang University for the help in the mass-spectrometry data analysis. This work is supported by the high technology development program of China (863) (grant no. 2008AA092602).
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