Research article | Open | Published:
Synaptotagmin gene content of the sequenced genomes
BMC Genomicsvolume 5, Article number: 43 (2004)
Synaptotagmins exist as a large gene family in mammals. There is much interest in the function of certain family members which act crucially in the regulated synaptic vesicle exocytosis required for efficient neurotransmission. Knowledge of the functions of other family members is relatively poor and the presence of Synaptotagmin genes in plants indicates a role for the family as a whole which is wider than neurotransmission. Identification of the Synaptotagmin genes within completely sequenced genomes can provide the entire Synaptotagmin gene complement of each sequenced organism. Defining the detailed structures of all the Synaptotagmin genes and their encoded products can provide a useful resource for functional studies and a deeper understanding of the evolution of the gene family. The current rapid increase in the number of sequenced genomes from different branches of the tree of life, together with the public deposition of evolutionarily diverse transcript sequences make such studies worthwhile.
I have compiled a detailed list of the Synaptotagmin genes of Caenorhabditis, Anopheles, Drosophila, Ciona, Danio, Fugu, Mus, Homo, Arabidopsis and Oryza by examining genomic and transcript sequences from public sequence databases together with some transcript sequences obtained by cDNA library screening and RT-PCR. I have compared all of the genes and investigated the relationship between plant Synaptotagmins and their non-Synaptotagmin counterparts.
I have identified and compared 98 Synaptotagmin genes from 10 sequenced genomes. Detailed comparison of transcript sequences reveals abundant and complex variation in Synaptotagmin gene expression and indicates the presence of Synaptotagmin genes in all animals and land plants. Amino acid sequence comparisons indicate patterns of conservation and diversity in function. Phylogenetic analysis shows the origin of Synaptotagmins in multicellular eukaryotes and their great diversification in animals. Synaptotagmins occur in land plants and animals in combinations of 4–16 in different species. The detailed delineation of the Synaptotagmin genes presented here, will allow easier identification of Synaptotagmins in future. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies.
Synaptotagmin (Syt) 1 was initially found as a protein component of synaptic vesicles . New members of the Syt gene family have subsequently been discovered by DNA sequence similarity [2–15]. Syts encode proteins which share a common structure: an N-terminal transmembrane sequence joined to a variable length linker, followed by two tandemly arranged, distinct C2 domains, C2A and C2B. At present, a great deal more is known about Syt1 than the other Syts because it functions crucially in synaptic vesicle trafficking in the nervous systems of animals . Other Syts are implicated in trafficking events in the nervous system as well as in various other tissues [17, 18]. Certain Syts are known to express alternatively spliced transcripts [19–21] and RNA editing of Drosophila Syt1 has been described . Little is known however, about the details of the variations in expression of different Syts.
Public sequence database resources are becoming quite comprehensive, including vast numbers of transcript sequences from a wide variety of organisms as well as a number of relatively complete genome sequences. Systematic identification of Syts by database searching makes it possible to begin to address questions such as: what is the evolutionary extent of this gene family? where do these genes appear on the tree of life? and how many of these genes does an organism need?
Building on my previous effort to extract the Syt content of the sequenced genomes  I have now collected information for 98 Syts from organisms with sequenced genomes. Transcript sequences reveal abundant variation in Syt expression and indicate the presence of Syts in all land plants and all animals.
Results and Discussion
Identification of Syts
Previously  I used a 44 amino acid sequence probe, representing the most highly conserved stretch of all the known Syts, and lying within a single exon in the C2B region, to search the sequence databases. This probe detected all the loci within the available genomes which could harbour Syts, but in order to confirm that these loci did indeed encode Syts it was necessary to ascertain that all the relevant parts were present (N-terminal transmembrane sequence, variable length linker, C2A and C2B). Whilst some regions (C2A and C2B) are well conserved, there is great variation in the sequences of other regions. It is difficult to predict exons accurately from genomic sequence unless a good degree of sequence similarity is present. Transcript sequences can reveal the true gene structure but few transcripts were available at that time, so although I could locate the already known Syts in Caenorhabditis, Drosophila and Homo, it was clear that there were more potential Syts in each of these genomes and that Syt relatives may even be present in plants, which would indicate a general function for this gene family, not restricted to the operation of nervous systems.
Recently, more genomes have been sequenced and some very good transcript resources have become available. I have also carried out cDNA library screening and RT-PCR to investigate the Arabidopsis Syts, the novel Homo Syts and the alternative splicing of Rattus Syt1 (accession numbers aj617615-aj617630). I used tblastn and blastn to search sequences at NCBI , EBI , Ensembl  and JGI . I assembled transcript sequences into gap4 databases  and used Spin  and Align  to compare transcripts with genomic sequence. I have compiled a list of 98 Syts from the genomes of Caenorhabditis, Anopheles, Drosophila, Ciona, Danio, Fugu, Mus, Homo, Arabidopsis and Oryza ([see Additional File 1] entries 1–98). This list summarizes the results of the database searches and includes genomic locations, amino acid sequences, exon structures and alternative splicing patterns. The identities of all the sequences examined here are summarized in Table 1. Where Syt synonyms exist, I have chosen the human gene names used by Ensembl  but have also indicated synonyms within parentheses.
Fig. 1 shows the chromosomal locations of Homo and Mus Syts. Syt2 and Syt14, Syt6 and Syt11, Syt8 and Syt9, Syt3 and Syt5, and Syt7 and Syt12, are linked in both Homo and Mus. Syt4, Syt15, and Syt16 are each solitary in both Homo and Mus. Linkage of Syt1, Syt10 and Syt13 is different in Homo and Mus. Different Homo (Syt9) and Mus (Syt4, Syt12) Syts are associated with overlapping antisense transcripts.
I used clustalw at EBI  to compare all 98 Syts (fig. 2) and Multalin  followed by manual editing to produce mulitple alignments (figs. 3,4,5,6,7,8). Where alternative splicing produces complex sequence variation, I chose one representative sequence. Fig. 2 shows the clustalw cladogram tree of relationships between the Syts. The multiple alignments are arranged in the same way, with N-terminus and linker regions in figs 3,4,5 and C2A to C-terminus regions in figs 6,7,8. Intron positions, alternative splicing and RNA-edited positions are indicated.
Animal Syts are distributed over more than 7 main branches of the cladogram tree while plant Syts occupy a separate main branch. Groups of orthologues and paralogues appear on closely linked sub-branches. A group of orthologues includes genes from different species for example, all Syt1 genes. Paralogues are multiple versions of one gene within the same species. The paralogues of Syt1 in Mus and Homo are Syt2, Syt5 and Syt8. I have used the tree and multiple alignment information to give provisional names to as many Syts as possible (Table 1).
The 6 Arabidopsis Syts and 8 Oryza Syts are each found on three sub-branches. The Oryza genome is polyploid so one would expect multiple copies of many genes, and since the genome sequence is incomplete, further Oryza Syts may yet be found. Searches of plant transcript sequences, reveal the presence of Syts in all the land plants. Sequences from Pinus, Physcomitrella and Ceratopteris ([see Additional File 1] entries 99–107) demonstrate the presence of plant Syts across the whole evolutionary range of land plants.
Animals have a more diverse array: 7 Syts in Caenorhabditis, 5 or more in Anopheles (incomplete genome sequence), 7 in Drosophila, 4 or more in Ciona (a surprisingly small number perhaps, but an incomplete genome sequence), 13–14 in Danio and Fugu (incomplete genome sequences) and 16 in Mus and Homo. Bearing in mind that some of the genome sequences are incomplete, the overall picture appears to reflect both acquisition and loss of different types of Syt, with different animals bearing different arrays of Syts. I have highlighted a motif (G X X X P E L Y) in the linker region of the Syt15 orthologues (fig. 4) which is shared with the otherwise unrelated, vertebrate specific branch of Syts which includes Syt9, Syt10, Syt6 and Syt3. Such a conserved motif probably indicates the specification of a common function.
Expression of variant Syts
Alternative splicing (see Additional File 1) adds a further level of diversity to Syts. The large numbers of Mus and Homo transcripts in particular, show abundant alternative splicing which involves coding regions as well as both upstream and downstream regions. There are common patterns of alternative splicing in Mus and Homo as well as species specific patterns. For example, both Mus and Homo Syt11 transcripts, use atypical GC intron donors in the final intron, rather than the typical GT donors which are present, to specify a change in the second calcium coordinating position in the C2B region (fig. 7). In fish, the same sequence is encoded via typical intron donors. Another such example is Syt16 where both Mus and Homo use atypical GC intron donors in the final intron preceding the C2A region, but Fugu Syt16 does not. There are numerous examples of differences in the patterns of alternative splicing between Mus and Homo. Certain regions of the coding sequences are altered in specific Syts but overall, these regions range from N-terminus to C-terminus indicating a sophisticated control of many functions.
Examples of common patterns of sequence variation in certain Syts include the alternative splicing of the short linker of Syt1 in Anopheles, Drosophila, Mus and Homo (fig. 3). The functional consequences of this alternative splicing have recently been investigated . In Syt1, the C2B region undergoes alternative splicing in Caenorhabditis and RNA editing in Anopheles and Drosophila. Alternative splicing equivalent to that of Caenorhabditis has just also been described in Aplysia . There is no evidence for equivalent alteration of Ciona, Danio, Fugu, Mus or Homo Syt1. It is intriguing to note that this region in the most abundantly expressed Arabidopsis Syt is also encoded by alternative exons. Alterations of the N-terminal end of Syt6 and the C-terminal ends of many Syts in the same vertebrate specific branch, as well as variable insertions into the linker region of Mus and Homo Syt7 (although nothing similar is found in other Syt7 orthologues) and insertions into the C2B region of the Syt14 homologues are further examples of common patterns of sequence variation in certain Syts. The true complexity of Syt alternative splicing needs to be examined systematically in detail.
It is fortunate that the transcript sequencing projects in Mus and Homo have generated sequences from many different cell types at different stages of development, as it is likely that the production of variant Syts is under cell type and temporal control. Alternative splicing of exons in the 5' untranslated (UTS) region of Syt1 in mammals (see Additional File 1 and accession numbers aj617615-aj617619 for alternative splicing in Homo, Mus and Rattus) seems to be particularly complex and is the likely explanation for the described variations . This was not seen in the original 5' mapping work  but RNase protection (RPA) analysis in R. norvegicus and R. rattus (fig. 9) confirms the evidence of complex, species specific alternative splicing in this region of Syt1 in the sequence databases. Alternative splicing of this region is also evident in Ciona Syt1 and a functional analysis of this region in the related organism Halocynthia has recently been carried out . Insufficient transcript evidence is currently available from other organisms to establish the universality of Syt1 5'UTS alternative splicing.
I have described more than 98 Syts from a broad range of animals and plants. Much remains to be done to understand the control of the expression and location of the range of variants produced by each Syt. There is no evidence of Syts in single cell organisms or those with the most simple forms of multicellularity (algae, fungi, slime moulds) leading one to speculate that these genes may be necessary for communication in more differentiated cell systems. Although C2 domains are present in the simpler eukaryotes, the distinctly conserved C2A-C2B arrangement is unique to Syts. All of the plant Syts share the transmembrane-linker-C2A region with a family of genes which encode proteins with variable numbers of C2 domains. This family has members in yeast, fungi, metazoa, land plants and trypanosoma, but there is no evidence of family members in other eukaryotes at present. The first functional analysis of the yeast members (tricalbins) has just been published  but the family is poorly characterized otherwise. Additional File 1 entries 108–126 describe the non-Syt members of this gene family in Arabidopsis, Oryza, Caenorhabditis, Drosophila, Homo, Saccharomyces and Trypanosoma. The analogous situation is not found in animals, where the relation between Syts and other gene families is restricted to C2 domain sequence similarity. The clustalw cladogram tree of all the sequences described in this paper is shown in fig. 10.
The advantages of performing an evolutionary analysis of Syts and attempting to understand their origins and diversity include the possibility of exhaustively defining the functions of a minimal set in a model organism (eg. Arabidopsis, Ciona). Comparative analysis of subgroups of Syts from a range of evolutionary lineages helps to define exactly which sequences are required to maintain function and which are able to diversify (see  for a structural evolutionary analysis of the C2 domains of Syts). The patterns of alternative splicing displayed by certain groups of Syts indicate enormous functional diversity that is only beginning to be understood. It will be fascinating to discover what it is about certain animal Syts that distinguishes them as essential players in neurotransmission.
RT-PCR and cDNA library screening
RT-PCR from Rattus brain mRNA was carried out with Pfu-turbo polymerase. A Homo brain cDNA library (Clontech) was screened with probes for the 6 novel human loci identified in  (accession numbers aj303363-aj303368). An Arabidopsis whole plant cDNA library (Stratagene) was screened with probes for the loci identified in . The probes were produced by PCR from genomic DNA which was a gift from Ian Furner (Cambridge University department of Genetics).
RNase protection analysis
RNase protection analysis (RPA) analysis was carried out as described . Rattus rattus brain was a gift from S.Redrobe at Bristol Zoo. Brain mRNA was prepared from Rattus rattus and from Rattus norvegicus (Sprague-Dawley) by guanidine isothiocyanate followed by polyA selection with oligo-dT cellulose. Regions of the 5' untranslated (5'UTS) portion of sequence accession x52772 (Rattus rattus Syt1) were cloned using RT-PCR with Rattus brain mRNA. RPA probes were produced using the Maxiscript kit (Ambion) from pBSIIKS- clones containing insert sequences aj617620-aj617622.
Perin MS, Fried VA, Mignery GA, Jahn R, Südhof TC: Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990, 345: 260-263. 10.1038/345260a0.
Geppert M, Archer BT, Südhof TC: Synaptotagmin II a novel differentially distributed form of synaptotagmin. J Biol Chem. 1991, 266: 13548-13552.
Mizuta M, Inagaki N, Nemoto Y, Matsukura S, Takahashi M, Seino S: Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells. J Biol Chem. 1994, 269: 11675-11678.
Hillbush BS, Morgan JI: A third synaptotagmin gene, Syt3, in the mouse. Proc Natl Acad Sci. 1994, 91: 8195-8199.
Craxton M, Goedert M: Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain. FEBS Lett. 1995, 361: 196-200. 10.1016/0014-5793(95)00176-A.
Hudson AW, Birnbaum MJ: Identification of a nonneuronal isoform of synaptotagmin. Proc Natl Acad Sci. 1995, 92: 5895-5899.
Li C, Ullrich B, Zhang JZ, Anderson RGW, Brose N, Südhof TC: Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 1995, 375: 594-599. 10.1038/375594a0.
Babity JM, Armstrong JN, Plumier J-CL, Currie RW, Robertson HA: A novel seizure-induced synaptotagmin gene identified by differential display. Proc Natl Acad Sci. 1997, 94: 2638-2641. 10.1073/pnas.94.6.2638.
Thompson CC: Thyroid hormone-responsive genes in developing cerebellum include a novel synaptotagmin and a hairless homolog. J Neurosci. 1996, 16: 7832-7840.
von Poser C, Ichtchenko K, Shao X, Rizo J, Südhof TC: The evolutionary pressure to inactivate a subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding. J Biol Chem. 1997, 272: 14314-14319. 10.1074/jbc.272.22.14314.
von Poser C, Südhof TC: Synaptotagmin 13: structure and expression of a novel synaptotagmin. Eur J Cell Biol. 2001, 80: 41-47.
Fukuda M, Mikoshiba K: Characterization of KIAA1427 protein as an atypical synaptotagmin (SytXIII). Biochem J. 2001, 354: 249-257. 10.1042/0264-6021:3540249.
Craxton M: Genomic analysis of synaptotagmin genes. Genomics. 2001, 77: 43-49. 10.1006/geno.2001.6619.
Fukuda M: Molecular cloning, expression, and characterization of a novel class of synaptotagmin (SytXIV) conserved from Drosophila to humans. J Biochem. 2003, 133: 641-649. 10.1093/jb/mvg082.
Fukuda M: Molecular cloning and characterization of human, rat, and mouse synaptotagmin XV. Biochem Biophys Res Commun. 2003, 306: 64-71. 10.1016/S0006-291X(03)00911-2.
Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Südhof TC: Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 1994, 79: 717-727.
Südhof TC: Synaptotagmins: why so many?. J Biol Chem. 2002, 277: 7629-7632. 10.1074/jbc.R100052200.
Tucker WC, Chapman ER: Role of synaptotagmin in Ca2+ -triggered exocytosis. Biochem J. 2002, 366: 1-13.
Fukuda M, Mikoshiba K: A novel alternatively spliced variant of synaptotagmin VI lacking a transmembrane domain. J Biol Chem. 1999, 274: 31428-31434. 10.1074/jbc.274.44.31428.
Craxton M, Goedert M: Alternative splicing of synaptotagmins involving transmembrane exon skipping. FEBS Lett. 1999, 460: 417-422. 10.1016/S0014-5793(99)01382-4.
Sugita S, Han W, Butz S, Liu X, Fernandez-Chacon R, Lao Y, Südhof TC: Synaptotagmin VII as a plasma membrane Ca2+ sensor in exocytosis. Neuron. 2001, 30: 459-473. 10.1016/S0896-6273(01)00290-2.
Hoopengardner B, Bhalla T, Staber C, Reenan R: Nervous system targets of RNA editing identified by comparative genomics. Science. 2003, 301: 832-836. 10.1126/science.1086763.
National Center for Biotechnology Information. [http://www.ncbi.nlm.nih.gov]
European Bioinformatics Institue. [http://www.ebi.ac.uk]
Ensembl Genome Browser. [http://www.ensembl.org]
Joint Genome Initiative Ciona intestinalis v1.0. [http://genome.jgi-psf.org/ciona4/ciona4.home.html]
Staden Package WWW site. [http://www.mrc-lmb.cam.ac.uk/pubseq/staden_home.html]
Pearson WR: In Methods in Molecular Biology, Computer analysis of sequence data, PartII. Edited by: Griffin AM, Griffin HG. 1994, New Jersey: Humana Press, 365-389.
Corpet F: Multiple sequence alignment with hierarchical clustering. Nuc Acids Res. 1988, 16: 10881-10890.
Nakhost A, Houeland G, Castelluci VF, Sossin W: Differential regulation of transmitter release by alternatively spliced froms of synaptotagmin 1. J Neurosci. 2003, 23: 6238-6244.
Nakhost A, Houeland G, Blandford VE, Castelluci VF, Sossin W: Identification and characterization of a novel C2B splice variant of synaptotagmin I. J Neurochem. 2004, 89: 354-363.
Bagalá C, Kolev V, Mandinova A, Soldi R, Mouta C, Graziani I, Prudovsky I, Macaig T: The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1. Biochem Biophys Res Commun. 2003, 310: 1041-1047. 10.1016/j.bbrc.2003.09.119.
Perin MS, Brose N, Jahn R, Südhof TC: Domain structure of synaptotagmin (p65). J Biol Chem. 1991, 266: 623-629.
Katsuyama Y, Matsumoto J, Okada T, Ohtsuka Y, Chen L, Okado H, Okamura Y: Regulation of synaptotagmin gene expression during ascidian embryogenesis. Dev Biol. 2002, 244: 293-304. 10.1006/dbio.2002.0584.
Schulz TA, Creutz CE: The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family. Biochemistry. 2004, 43: 3987-3995. 10.1021/bi036082w.
Jiménez JL, Smith GR, Contreras-Moreira B, Sgouros JG, Meunier FA, Bates PA, Schiavo G: Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches. J Mol Biol. 2003, 333: 621-639. 10.1016/j.jmb.2003.08.052.
Sutton RB, Davletov BA, Berghuis AM, Südhof TC, Sprang SR: Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell. 1995, 80: 929-938.
Sutton RB, Ernst JA, Brunger AT: Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction. J Cell Biol. 1999, 147: 589-598. 10.1083/jcb.147.3.589.
I wish to thank Nicola Ramsey for galvanising discussions.