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Integrative and Comparative Biology, volume 50, number 1, pp. 86–97 doi:10.1093/icb/icq010 SYMPOSIUM Genomics Reveal Ancient Forms of Stanniocalcin in Amphioxus and Tunicate Graeme J. Roch and Nancy M. Sherwood1, *Department of Biology, University of Victoria, Victoria, British Columbia V8W 3N5, Canada From the symposium ‘‘Insights of Early Chordate Genomics: Endocrinology and Development in Amphioxus, Tunicates and Lampreys’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2010, at Seattle, Washington. 1 E-mail: [email protected] Synopsis Stanniocalcin (STC) is present throughout vertebrates, including humans, but a structure for STC has not been identified in animals that evolved before bony fish. The origin of this pleiotropic hormone known to regulate calcium is not clear. In the present study, we have cloned three stanniocalcins from two invertebrates, the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae. Both species are protochordates with the tunicates as the closest living relatives to vertebrates. Amphioxus are basal to both tunicates and vertebrates. The genes and predicted proteins of tunicate and amphioxus share several key structural features found in all previously described homologs. Both the invertebrate and vertebrate genes have four conserved exons. The predicted length of the single pro-STC in Ciona is 237 amino acids and the two pro-hormones in amphioxus are 207 and 210 residues, which is shorter than human pro-STCs at 247 and 302 residues due to expansion of the C-terminal region in vertebrate forms. The conserved pattern of 10 cysteines in all chordate STCs is crucial for identification as amphioxus and tunicate amino acids are only 14–23% identical with human STC1 and STC2. The 11th cysteine, which is the cysteine shown to form a homodimer in vertebrates, is present only in amphioxus STCa, but not in amphioxus STCb or tunicate STC, suggesting the latter two are monomers. The expression of stanniocalcin in Ciona is widespread as shown by RT-PCR and by quantitative PCR. The latter method shows that the highest amount of STC mRNA is in the heart with lower amounts in the neural complex, branchial basket, and endostyle. A widespread distribution is present also in mammals and fish for both STC1 and STC2. Stanniocalcin is a presumptive regulator of calcium in both Ciona and amphioxus, although the structure of a STC receptor remains to be identified in any organism. Our data suggest that amphioxus STCa is most similar to the common ancestor of vertebrate STCs because it has an 11th cysteine necessary for dimerization, an N-glycosylation motif, although not the canonical one in vertebrate STCs, and similar gene organization. Tunicate and amphioxus STCs are more similar in structure to vertebrate STC1 than to vertebrate STC2. The unique features of STC2, including 14 instead of 11 cysteines and a cluster of histidines in the C-terminal region, appear to be found exclusively in vertebrates. Introduction Stanniocalcin is a large protein hormone that was originally purified from specialized glands, known as the corpuscles of Stannius, associated with the kidneys of teleostean and holostean fishes. Wagner et al. (1986) isolated and provided a partial aminoacid sequence for the first stanniocalcin (referred to as ‘teleocalcin’) from sockeye salmon corpuscles. They determined that it was a dimerized glycoprotein with hypocalcemic activity. Shortly thereafter, an orthologous hormone was isolated, cloned, and sequenced in eels (Butkus et al. 1987). Characterization of the hormone in several additional teleosts followed, including other salmonids; trout (Lafeber et al. 1988a; Lafeber et al. 1988b), chum (Sundell et al. 1992; Yamashita et al. 1995) and coho salmon (Wagner et al. 1988; Wagner et al. 1992), supporting the initial evidence of calcium downregulation by secretion of stanniocalcin from the corpuscles to effectors in the gills. More recently, the hormone was isolated from divergent groups of bony fishes, including the early teleostean bony-tongued fishes: arawana, butterflyfish, and Advanced Access publication April 1, 2010 ß The Author 2010. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected]. 87 Stanniocalcin in amphioxus and tunicate elephantnose (Amemiya et al. 2002; Amemiya et al. 2006), and the non-teleostean gar and bowfin (Amemiya and Youson 2004). All these hormones share consistent structural features, including a conserved N-glycosylation site and 10 invariant cysteine residues that form intrachain disulfide bonds. Also, an 11th conserved cysteine residue that is critical to dimerization (Hulova and Kawauchi 1999; Trindade et al. 2009) is found in all species with the exception of the bony-tongued fishes (Order, Osteoglossiformes). The latter secrete a unique, monomeric isoform of stanniocalcin (Amemiya et al. 2002; Amemiya et al. 2006). A human ortholog of stanniocalcin was later identified and cloned (Chang et al. 1995; Wagner et al. 1995; Olsen et al. 1996). Mouse stanniocalcin was also isolated (Chang et al. 1996), and the mammalian homologs both shared moderate (450%) amino-acid identity with their fish counterparts, as well as the conserved structural features. The expression of mammalian stanniocalcin was widespread throughout both endocrine and non-endocrine tissues, leading to the suggestion that it might serve a more autocrine or paracrine role (Ishibashi and Imai 2002). Early investigations of function for mammalian stanniocalcins suggested a familiar role in mineral homeostasis, although focused more on upregulated phosphate reabsorption rather than on downregulation of calcium (Haddad et al. 1996; Olsen et al. 1996). As research progressed, several novel roles for the hormone were implicated, including bone formation, growth effects, angiogenesis and, neuroprotection (Yoshiko and Aubin 2004). This was complicated by the discovery of tissue-specific isoforms of stanniocalcin found in the ovaries, adrenals, and adipocytes, referred to as ‘big stanniocalcin’ (Paciga et al. 2003). The nature of the big isoform is still unknown, even after studies on its composition and structure (Trindade et al. 2009). A second stanniocalcin gene was idenitified in mice and humans, and cloned by several groups (Chang and Reddel 1998; DiMattia et al. 1998; Ishibashi et al. 1998). This hormone, known as STC2, was also expressed across a wide variety of tissues and possessed the conserved structural features of the original stanniocalcin, now referred to as STC1. STC2 contained 3 additional conserved cysteines, and a histidine-rich C-terminal region. Later, STC2 was isolated from fishes (Luo et al. 2005; Shin and Sohn 2009) and birds, indicating that it was not a mammalian innovation. Functional activities of STC2 are not as highly defined as those of STC1, but recent work has demonstrated roles in the unfolded protein response (Ito et al. 2004), ovarian development (Luo et al. 2005), growth (Gagliardi et al. 2005), pancreatic alpha-cell function (Moore et al. 1999), and cancer (Bouras et al. 2002; Raulic et al. 2008; Ieta et al. 2009; Tamura et al. 2009). It is clear that the functional portfolio of the stanniocalcin family has expanded from its roots in early fish studies. Until now, the body of stanniocalcin research has focused on species within the vertebrates, with the most divergent isolated orthologs belonging to the basal Actinopterygii. Limited investigations of invertebrate stanniocalcins have been performed using fish antisera on sea snails (Wendelaar Bonga et al. 1989) and the horse leeches (Tanega et al. 2004). These results, although tantalizing, leave us without an isolated hormone that could be sequenced and confirmed. Questions regarding the origin of the stanniocalcins have thus remained largely unanswered beyond the bony fishes. Recent developments in the genomics of two model invertebrate chordates (protochordates), the tunicate Ciona intestinalis (Dehal et al. 2002) and the amphioxus Branchiostoma floridae (Putnam et al. 2008) have provided key insights into the evolution of vertebrates. Both animals have genomes that possess simplified versions of several multi-membered vertebrate gene families and, by comparison, inferences regarding the ancestral chordate complement may be drawn. Utilizing genomic resources, we isolated and cloned a single stanniocalcin ortholog from C. intestinalis and 2 from B. floridae. These orthologs possess key structural features conserved in their vertebrate counterparts. Phylogenetic analysis suggests that the protochordate stanniocalcins are monophyletic and distinct from both vertebrate STC1 and STC2. Together with expression studies, our data support the presence of invertebrate stanniocalcins as part of a hormone family of ancient mediators of mineral homeostasis, dating at least to the origin of the chordates. Methods Animals Wild-type adult C. intestinalis individuals were provided by the Ascidian Stock Center (Santa Barbara, CA, USA). Animals were kept in seawater until dissection, after which their tissues were snap frozen in liquid nitrogen, and stored at 808C. Wild-type B. floridae adults were purchased from Gulf Specimen Marine Laboratory (Panacea, FL, USA). Whole animals were snap-frozen in liquid nitrogen and stored at 808C. 88 RNA extraction and cDNA synthesis Total ribonucleic acid (RNA) was extracted from individual C. intestinalis tissues and whole B. floridae adults. Tissues were immersed in lysis buffer and processed using the Qiagen RNeasy Mini Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer’s instructions. Extracted RNA was quantitated by absorbance at 260 nm, and 1 mg samples were reverse-transcribed with Superscript III (Invitrogen Canada Inc., Burlington, ON, Canada) as per the manufacturer’s guidelines, using 200 units of enzyme and 500 ng oligo dT(12– 18) to amplify messenger (m) RNAs. The reaction was incubated at 508C for 60 min. 50 and 30 rapid amplification of cDNA ends (RACE)-ready complementary deoxyribonucleic acid (cDNA) was also prepared from C. intestinalis tissues with the RLM-RACE kit (Applied Biosystems/Ambion, Austin, TX, USA) using 1 mg total RNA and the manufacturer’s standard protocol. PCR amplification and cloning From genomic resources at the Joint Genome Institute (JGI) for C. intestinalis (http://genome .jgi-psf.org/Cioin2/Cioin2.home.html) and B. floridae (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html), full or partial stanniocalcin gene models were identified and primers were designed against these sequences. Primers for full-length, open-reading frames were designed as follows: C. intestinatlis stc forward 50 -ATGTACGCTACAAGACATTGTAC CG-30 , reverse 50 -TTAGCTCTGTCTTGGCGAA 0 CT-3 ; B. floridae stca forward 50 -ATGACAGCAGT GGGAAGACC-30 , reverse 50 -TTAGCCGTACATGGA GAACC-30 , B. floridae stcb 50 -ATGCAGTTTGTAAGC TGGATGC-30 , reverse 50 -TCACTTGCCGAGGCCCC TCA-30 . Sequences were amplified from cDNA samples using 1 unit of Platinum Taq DNA Polymerase High Fidelity (Invitrogen), with 2 mM magnesium sulfate, 0.2 mM mixed deoxynucleoside triphosphates (dNTPs), and 0.4 mM of each primer. Cycling conditions were as follows: 948C for 2 min, followed by 35 cycles of 948C for 15 s, then 55–608C for 30 s, and 688C for 2 min. Polymerase chain reaction (PCR) for 50 and 30 RACE was carried out using RLM-RACE kit primers, and the respective forward and reverse primers from C. intestinalis previously mentioned. Amplicons were separated on a 1.2% agarose gel, and bands were excised and purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. Purified amplicons were ligated to the pGEM-T vector (Promega Corporation, Madison, WI, USA) overnight at 168C G. J. Roch and N. M. Sherwood and transformed by electroporation into XL-1 Blue electrocompetent cells (Stratagene, La Jolla, CA, USA). Cells were plated under ampicillin selection and incubated overnight at 378C, and colonies were picked and placed in Lysogency broth (LB) overnight at 378C. Plasmid DNA was extracted from broth cultures using the QuickClean 5M Miniprep Kit (Genscript USA Inc., Piscataway, NJ, USA) using the manufacturer’s protocol. Plasmids were separated on 1.2% agarose gels, and clones were sequenced at the UVic CBR DNA Sequencing Facility (Victoria, BC, Canada). Phylogenetics Predicted full-length peptide sequences from C. intestinalis and B. floridae were aligned with stanniocalcin orthologs from human and zebra fish listed in the National Center for Biotechnology Information (NCBI) Protein database (http://www.ncbi.nlm.nih .gov/protein). Human STC1 (NCBI accession NP_003146.1), human STC2 (NP_003705.1), zebra fish STC1 (NP_956833.1), and zebra fish STC2 (NP_001014827.1) were aligned with protochordate sequences using ClustalW (Thompson et al. 2002). Gene diagrams were designed using the JGI genomic resources for C. intestinalis and B. floridae, as previously mentioned. Additional information on sequences for the untranslated regions of amphioxus stcb was procured from the B. floridae cDNA Database (http://amphioxus.icob.sinica.edu.tw/). The human STC gene diagram was based on information located within NCBI’s Gene database (http:// www.ncbi.nlm.nih.gov/gene). An amino-acid identity matrix was created using sequences lacking their putative signal peptides in BioEdit (Hall 1999). A phylogenetic tree based on maximum likelihood was produced using the previously mentioned peptide sequences as well as the following, listed with their respective NCBI accession numbers: silver arawana STC1 (BAB43868.1), bowfin STC1 (BAC66163.1), butterflyfish STC1 (BAD99601.1), chicken STC1 (XP_425760.2), chicken STC2 (XP_414534.2), eel STC1 (AAB91483.1), elephantnose STC1 (BAD99600.1), flounder STC1 (ABI64157.1), flounder STC2 (ACJ06521.1), frog STC1 (NP_001086522.1), frog STC2 (NP_001016004.1), fugu STC1 (NP_001072056.1), fugu STC2 (NP_001072057.1), gar STC1 (BAC66164.1), mouse STC1 (NP_ 033311.3), mouse STC2 (NP_035621.1), opossum STC1 (XP_001373050.1), opossum STC2 (XP_ 001370619.1), platypus STC1 (XP_001506746.1), and salmon STC1 (AAB26419.1). Sequences were aligned in ClustalW, trimmed before and after the region 89 Stanniocalcin in amphioxus and tunicate containing conserved cysteines, and degapped. Trees were generated in PhyML 3.0 (Guindon et al. 2009) using the LG (Le and Gascuel amino-acid replacement matrix) substitution model with the following options: proportion of invariable sites–estimated, tree topology search operations–best of NNI and SPR, and bootstrapping with 100 replicates. Reverse transcriptase and quantitative PCR of stanniocalcin in C. intestinalis tissues Individual tissue cDNA samples were analyzed for relative stc expression by standard reverse transcriptase-PCR (RT-PCR) using the following primers: forward 50 -GTTTACGAAACAGTCACTGCGATG-30 and reverse 50 -TTGACATAGCAGCCCGACAAAC-30 . Amplification was carried out using 2.5 units of Taq DNA Polymerase (Invitrogen) with 2 mM magnesium chloride, 0.2 mM mixed dNTPs, and 0.4 mM of each primer under the following conditions: 948C for 3 min, followed by 33 cycles of 948C for 30 s, then 558C for 30 s, and 728C for 45 s. Reactions were loaded on a 1.2% agarose gel and electrophoresed, stained with ethidium bromide and imaged. Quantitative PCR (qPCR) was carried out to determine relative stc expression in C. intestinalis as follows. Primers amplifying cDNAs for stanniocalcin and the housekeeping reference, TATA-box binding protein (TBP), were as follows: stc forward 50 -CG ACAATCCGCTGGTATC-30 , reverse 50 -CGCTGGAA ATCCGACTTGA-30 ; tbp forward 50 -CAGTCAGTTC TCAAGTTATGAGCC-30 , reverse 50 -ATATACTTTG GCACCTGTTAGAACTAC-30 . Genes were amplified from tissue cDNA samples from three adult C. intestinalis in a Mx3000P Real-Time PCR System (Stratagene) with 1 unit Platinum Taq DNA Polymerase (Invitrogen), 3 mM magnesium chloride, 0.25 mM mixed dNTPs, and 0.25 mM of each primer. Cycling conditions were as follows: 958C for 5 min, followed by 40 cycles of 958C for 30 s, then 558C for 60 s, and 728C for 30 s. This was followed by a dissociation melting curve control to ensure fluorescence was due to a single amplified product. Following amplification and cycle threshold (Ct) value determination for the transcripts in each tissue, results were averaged and plotted as the relative ratio of target copies to reference copies. Results Isolation, sequencing, and structure of stanniocalcin from tunicates and amphioxus Initially, BLAST homology searches were performed on the draft genome databases of both the tunicate C. intestinalis (http://genome.jgi-psf.org/Cioin2/ Cioin2.home.html) and amphioxus B. floridae (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html) using full-length vertebrate stanniocalcins as queries. One tunicate and two amphioxus orthologs were identified, and primers were designed based on these sequences. Full-length stanniocalcin open reading frames (ORFs) from both amphioxus and tunicates were amplified by PCR from cDNA libraries derived from at least two individuals of each species, and the resulting amplicons were sequenced. The predicted full-length amino-acid sequences of the tunicate and amphioxus stanniocalcins are aligned in Fig. 1A with human and zebra fish STC1 and STC2 homologs. Conserved cysteine residues are highlighted in black and the predicted signal peptides are shaded gray. As shown, 10 of the 11 invariant cysteine residues within the vertebrate stanniocalcins were preserved in the protochordate orthologs as well. The 11th conserved cysteine residue in vertebrate stanniocalcin homologs, established as the site of disulfide linkage between stanniocalcin monomers, was only present in amphioxus STCa. Although the conserved N-glycosylation motif of vertebrate stanniocalcins, boxed in Fig. 1A, was absent from all of the protochordate sequences, a potential N-glycosylation motif at an alternate site was identified in amphioxus STCa using the N-glycosylation prediction algorithm from NetNGlyc 1.0 (http:// www.cbs.dtu.dk/services/NetNGlyc) (Fig. 1A). The C-terminal histidine-rich motif present in vertebrate STC2 homologs (shaded gray in Fig. 1A) was not present in either tunicate or amphioxus stanniocalcins. The putative gene structures of human STC1 and the protochordate stanniocalcins are displayed in Fig. 1B. As shown, both the tunicate and amphioxus orthologs have the conserved four-exon structure found in the vertebrates, indicating that it is preserved throughout the chordates. Phylogenetic analysis of chordate stanniocalcins The amino-acid identity of the tunicate and amphioxus mature stanniocalcins is compared with human and zebra fish homologs in Table 1. Sequences were aligned without signal peptides, and a matrix identifying identical amino acids between each pairwise comparison was generated. As shown, the human and zebra fish STC2 orthologs displayed the highest homology, at 60% identity. The next highest homology was found between the human and zebra fish STC1 orthologs, at 50% identity. The identity of the human and zebra fish STC1 and STC2 paralogs was significantly lower in any combination (21–26%). This was comparable with 90 G. J. Roch and N. M. Sherwood Fig. 1 (A) ClustalW alignment of putative stanniocalcin peptides from tunicates (C. intestinalis), amphioxus (B. floridae), as well as representative sequences for STC1 and STC2 from humans and zebra fish. The predicted signal peptides are highlighted at the N-terminus in gray, and conserved cysteine residues are highlighted in black. The C-terminal cysteine residue is also indicated as the site of dimerization. Predicted N-glycosylation sites are indicated and boxed, and the histidine-rich C-terminal motif in STC2 is highlighted in gray. (B) Gene diagrams for human and protochordate stanniocalcins. Exons are represented as boxes and introns as lines, with the shaded regions indicating the ORFs. Numbers correspond to the length of each exon in nucleotides. the identity of the two amphioxus paralogs at 24%. Tunicate stanniocalcin displayed low homology with both the vertebrate (17–23% identity) and the amphioxus (19–24% identity) sequences. Amphioxus STCa displayed slightly higher identity with the vertebrate homologs than with 91 Stanniocalcin in amphioxus and tunicate Table 1 Stanniocalcin amino-acid identity matrix Sequence Human STC1 Human STC1 (%) – Zebra fish STC1 (%) – Human STC2 (%) – Zebra fish STC2 (%) – Tunicate STC (%) – Amphioxus STCa (%) – Zebra fish STC1 50 – – – – – Human STC2 24 21 – – – – Zebra fish STC2 26 22 60 – – – Tunicate STC 23 21 17 17 – – Amphioxus STCa 23 23 17 18 24 – Amphioxus STCb 18 17 14 15 19 24 Predicted peptide sequences from tunicates (C. intestinalis), amphioxus (B. floridae), humans, and zebra fish were aligned without signal peptides using ClustalW. An identity matrix was computed with BioEdit Sequence Alignment Editor. Amino-acid identity percentages between all pairwise comparisons are listed. Fig. 2 Phylogenetic tree of stanniocalcin sequences. Protochordate protein sequences, along with vertebrate sequences from both STC1 and STC2 isoforms were aligned using the conserved region between cysteine residues. Maximum likelihood (PhyML) was used to construct the resulting phylogeny. Bootstrap analysis (100 bootstraps) was employed and the frequency of support is indicated at each node. STCb: 17–23% compared with 14–18%. Amphioxus STCa was also more homologous to tunicate STC; 24% identity versus 19% for STCb. Stanniocalcin protein sequences from representative species were aligned between the conserved cysteine residues and their phylogeny established using maximum likelihood (PhyML) with bootstrap support, as shown in Fig. 2. The vertebrate STC1 and STC2 proteins were grouped together in distinct clades. Further nodes established the monophyletic distinctions between the tetrapod and fish groups of STC2; however, this resolution was not repeated within the STC1 grouping. STC1 sequences within the fishes formed distinct groups corresponding to their phylogeny, with the non-teleosts (gar and bowfin) separate from the teleosts. Within the teleosts the bony-tongued Osteoglossiformes, including elephantnose, freshwater butterflyfish, and silver arawana, formed a clade separate from the others. It should be noted that the Osteoglossiformes also lack the terminal cysteine 92 Fig. 3 RT-PCR analysis of C. intestinalis stanniocalcin was performed for the indicated tissues using -tubulin as a control. These results were refined using qPCR, which shows the ratio of expressed stanniocalcin cDNA against a housekeeping control, TBP cDNA. residue that all other vertebrate stanniocalcins possess, critical to dimerization. The amphioxus and tunicate stanniocalcins were grouped together, separated from the vertebrate hormones. As seen in Fig. 2, the protochordate STCs were all highly divergent from vertebrate STC1s and STC2s, as well as from each other, with amphioxus STCb being significantly more divergent than the others. Expression of stanniocalcin in tunicates Expression localization studies on C. intestinalis stanniocalcin cDNA were carried out for a variety of tissues. As seen in Fig. 3, initial RT-PCR experiments indicated a high level of expression in a variety of tissues, most prominently the neural complex, heart, and gonad. When qPCR experiments were normalized against a housekeeping gene (Fig. 3), however, expression in the heart was predominant. Relative expression in the branchial basket, neural complex, and endostyle was also measurably higher than in other tissues tested. Discussion The sequences characterized in this study represent the first stanniocalcin gene orthologs cloned and G. J. Roch and N. M. Sherwood characterized from non-vertebrates. Previous research has identified stanniocalcins from several species of tetrapods and major groups of bony fishes (Fig. 2) including two species from non-teleost fish, the gar, and bowfin (Amemiya and Youson 2004). The only prior evidence of non-vertebrate stanniocalcin homologs came from experiments in which immunoreactivity was detected in sea-snail (Wendelaar Bonga et al. 1989) and leech tissues (Tanega et al. 2004) using fish antisera. In leeches, a salmon STC1 antibody and in situ hybridization with an RNA probe specific for salmon Stc1 resulted in staining within adipose and skin cells. Here, we present a single gene orthologous to vertebrate stanniocalcins from the tunicate C. intestinalis, and two orthologous genes from the amphioxus B. floridae. These invertebrate homologs possess conserved sequence elements found in their vertebrate counterparts, and combined with the pattern of stc gene expression displayed in tunicate they confirm a role for stanniocalcin dating back to the early chordates. The genes and predicted proteins of tunicate and amphioxus stanniocalcins share several key structural features found in all previously described homologs. The invertebrate genes have the conserved four-exon structure found in the vertebrates (Fig. 1B), although the invertebrate hormones are not as long as their vertebrate counterparts. Full-length tunicate pro-STC is predicted to be 237 amino acids and the amphioxus paralogs are between 207 and 210 amino acids, whereas human pro-STC1 and pro-STC2 are 247 and 302 amino acids, respectively. This is due to the C-terminal expansions in the vertebrate stanniocalcins, shown in Fig. 1A, downstream of the terminal conserved cysteine residue. The functional significance of this is unclear, although a C-terminal fragment of eel stanniocalcin representing this region has been demonstrated to repress calcium influx on its own (Fenwick and Verbost 1993; Verbost and Fenwick 1995). Apart from this C-terminal extension, the invertebrate hormones share the salient characteristics found in vertebrate stanniocalcins, including a signal peptide followed by the 10 invariant cysteines distributed throughout the mature hormone (Fig. 1A). These conserved cysteine residues are a hallmark of all STC homologs isolated so far. They are critical to a series of specific intrachain disulfide bridges, which have been confirmed by studies on salmon STC1 (Hulova and Kawauchi 1999) and human STC1 (Trindade et al. 2009). Vertebrate STC2 orthologs have an additional three conserved cysteine residues that the STC1 group and the invertebrates lack; the function of these has been Stanniocalcin in amphioxus and tunicate suggested to involve additional intermolecular disulfide bonds (Luo et al. 2005). Another feature unique to the STC2s is the C-terminal histidine-rich motif, displayed in Fig. 1A. None of the invertebrate sequences have these STC2-specific attributes. The vast majority of vertebrate stanniocalcins also possess a C-terminal 11th conserved cysteine residue, with the exception of homologs from teleost fishes within the order Osteoglossiformes (Amemiya et al. 2002; Amemiya et al. 2006). This cysteine is distinct from the rest in its function, as it has been demonstrated to be the sole determinant of STC1 dimerization in salmon and humans (Hulova and Kawauchi 1999; Trindade et al. 2009). The dimerization of native and recombinant stanniocalcins has been established in several species using reducing and non-reducing SDS–PAGE analysis, including salmon STC1 (Wagner et al. 1986; Flik et al. 1990), human STC1 and STC2 (Moore et al. 1999; Luo et al. 2005), hamster STC2 (Moore et al. 1999), and turbot STC1 (Shin et al. 2006), among others. The structural analysis of cysteine residues within salmon and human STC1 confirmed the C-terminal cysteine as the site of dimerization (Hulova and Kawauchi 1999; Trindade et al. 2009). Also, the lack of the terminal cysteine residue within silver arawana revealed a monomeric form of the hormone under reducing and non-reducing experiments (Amemiya et al. 2002; Amemiya et al. 2006). Tunicate STC and amphioxus STCb do not have this dimerizationcritical cysteine, indicating that they are present as monomers (Fig. 1A). Amphioxus STCa, however, contains a cysteine at the same relative position as the C-terminal residue in the vertebrate homologs, suggesting it may be dimerized. The consequences of this dimerization are not understood. The last highly conserved feature present within vertebrate stanniocalcins is the N-glycosylation motif present between the third and fourth conserved cysteines in both STC1 (Asn-Ser-Thr) and STC2 (Asn-Asn-Ser) (Fig. 1A). This site is postulated to play a critical role in the glycosylation of the mature hormone, which is the native state of vertebrate stanniocalcins isolated to date, including eel STC1 (Butkus et al. 1987), trout STC1 (Lafeber et al. 1988a; Lafeber et al. 1988b), bowfin STC1 (Marra et al. 1992), human STC1 and STC2 (Wagner et al. 1995; Moore et al. 1999; Luo et al. 2005), arawana STC1 (Marra et al. 1995; Amemiya et al. 2002), and turbot STC1 (Shin et al. 2006). None of the invertebrate stanniocalcin sequences have this conserved motif at the same site, although a N-glycosylation motif at a different site is present 93 in amphioxus STCa (Asn-Asn-Thr) (Fig. 1A), indicating it may be a glycoprotein in its native state. Given the number of conserved features between the invertebrate and vertebrate stanniocalcins, it is interesting they retain such low sequence identity. As seen in Table 1, the only sequences with more than 50% homology were the human and zebra fish STC1 and STC2 orthologs, respectively. The vertebrate paralogs shared significantly reduced identity, between 21% and 26%. The tunicate and amphioxus stanniocalcins were comparably distinct from both the vertebrate hormones and each other, with an identity of only 14% between amphioxus STCb and human STC2. Evidently, outside the conserved sequence features previously mentioned, the primary sequence of stanniocalcins across the Phylum Chordata can vary to a large extent. Given the cross-activating potential of STC-immunoreactive extracts from animals as distinct as salmon and leeches (Tanega et al. 2004), the data indicate that the conserved disulfide bridging and possibly glycosylation are the major determinants in the binding and functioning of the hormones. Elucidation of the stanniocalcin binding to a receptor or ion channel will prove essential to this determination. RT-PCR and qPCR experiments were carried out on tunicate stc mRNA to determine its tissue localization. As shown in Fig. 3, after normalization against a housekeeping gene, the qPCR displayed predominant expression of stc in the C. intestinalis heart, with lower expression in the neural complex, branchial basket, and endostyle. The branchial basket houses and includes the gill slits of tunicates, and the endostyle is a mucus secreting gland found in protochordates and jawless fishes that shares homology with the vertebrate thyroid gland (Ogasawara et al. 1999; Ogasawara 2000). Tunicates do not have an organ homologous to the corpuscles of Stannius, an outgrowth from the kidney and originally thought to be the region of exclusive stanniocalcin expression in fishes; thus, no direct comparisons can be made. Following the isolation of stanniocalcin in humans and other mammals, however, many other regions of stanniocalcin expression have been found in both tetrapods and fishes. Stanniocalcins in tetrapods are reported to be expressed in the nervous system (Zhang et al. 1998; McCudden et al. 2001; Shin et al. 2006; Tseng et al. 2009) and the thyroid (Chang et al. 1995; Moore et al. 1999). Also, human STC1 and STC2 have been identified in human heart among several other tissues (Chang et al. 1995; DiMattia et al. 1998; Ishibashi et al. 1998; Moore et al. 1999). Stanniocalcins have also been identified in the heart of gar and bowfin (Amemiya and Youson 2004), flounder (Hang and 94 G. J. Roch and N. M. Sherwood Table 2 Hormones related to calcium homeostatsis in amphioxus (B. floridae), tunicates (C. intestinalis) and humans. Hormones Amphioxus H Tunicate R H Human R H STC 1 1 Calcitonin 4 5,6,7 Calcitonin Gene-related peptide (CGRP) Parathyroid hormone (PTH) R 2,3 5 4 6,7,8 9 10,11 PTH-related protein (PTHrP) Calcitriol (Vitamin D metabolite) References: (1) Present study, (2) http://www.ncbi.nlm.nih.gov/protein/NP_003146.1, (3) http://www.ncbi.nlm.nih.gov/protein/NP_003705.1, (4) Holland et al. 2009, (5) Sekiguchi et al. 2009, (6) Sherwood et al. 2006, (7) Cardoso et al. 2006, (8) Kamesh et al. 2008, (9) Schubert et al. 2008, (10) Dehal et al. 2002, and (11) Yagi et al. 2003. The identification of hormones (H) or their cognate receptors (R) is indicated by a black box. Balment 2005; Shin and Sohn 2009) and turbot (Shin et al. 2006). With predominant expression in the heart, the question of tunicate stanniocalcin function is intriguing. Most of the studies on hormonal functions thus far have focused on its canonical role as a hypocalcemic regulator in fish gills or the pleiotropic roles it plays in phosphate and calcium homeostasis in mammals (for reviews see Yoshiko and Aubin 2004; Wagner and DiMattia 2006). A few recent studies have examined heart expression. Jiang et al. (2000) found immunoreactivity of STC1 in mouse heart cells at embryonic Day 8.5, and a few days later predominantly in the myocardium of the atrium and ventricle, suggesting an early role in heart development. Embryonic expression of STC2 was also detected in developing chick hearts (Mittapalli et al. 2006). A study by Sheikh-Hamad et al. (2003) focused on the role of STC1 in patients with heart failure. They found upregulation of the hormone in the failing heart, and also demonstrated that addition of STC1 to cultured cardiomyocytes resulted in inhibition of calcium currents through L-channels and slowed the cells’ contractions. Another recent study found high-affinity binding sites for STC1 on red blood cells (James et al. 2005). Taken together and considering the important role of calcium in heart contractions, it is possible that tunicate stanniocalcin plays a role in the developing heart and its regulation thereafter. The identification of stanniocalcin homologs in tunicates and amphioxus provides additional insight into the evolution of the hormone family in addition to the calcium-regulatory system at large. The gene structure of the vertebrate and invertebrate hormones has remained largely unchanged; however, significant mutation has altered all but the most conserved residues of the amino-acid sequence. It is now clear that the duplication producing STC1 and STC2 is an innovation that occurred early in vertebrate evolution with the tunicate and amphioxus hormones phylogenetically distinct from both, as seen in Fig. 3. Whether or not this divergence is present within the cyclostomes remains in question, as an orthologous sequence could not be retrieved from the lamprey (Petromyzon marinus) draft genome to date. The two amphioxus paralogs appear to be a lineage-specific duplication as well, although this cannot be certain as they are located on separate scaffolds, reducing the likelihood of tandem duplication. Structurally, the invertebrate stanniocalcins do share more conserved features with the STC1 orthologs to date. Stanniocalcin in amphioxus and tunicate 95 In the context of calcium regulation as a whole, we now have a more complete picture stretching to the invertebrate chordates provided by the tunicate and amphioxus genomes. As seen in Table 2, calcitonin genes have been identified in both protochordates, first in B. floridae by ourselves (Holland et al. 2008) and more recently in C. intestinalis (Sekiguchi et al. 2009). Calcitonin gene-related peptide has not been detected. Orthologs of the parathyroid hormone receptor have also been identified in tunicates and amphioxus, but not their cognate ligands. A nuclear receptor belonging to the same family as the calcitriol (vitamin D) receptor has been identified in tunicates but not in amphioxus. Combined, we now have a patchwork of hormones that regulate calcium homeostasis to investigate the origin of calcium regulation in invertebrates. Cardoso JCR, Pinto VC, Vieira FA, Clark MS, Power DM. 2006. Evolution of secretin family GPCR members in the metazoan. BMC Evol Biol 6:108. Acknowledgments Fenwick JC, Verbost P. 1993. A C-terminal fragment of the hormone stanniocalcin is bioactive in eels. Gen Comp Endocrinol 91:337–43. We thank Ryan Liebscher for his help with an early phase of the research. 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