Conservation and divergence of ADAM family proteins in the Xenopus genome
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Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species.
Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region.
Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease activities are absent, while other novel ADAM proteins in this species are predicted by this study. The conservation and unique divergence of ADAM genes in Xenopus probably reflect the particular selective pressures these amphibian species faced during evolution.
KeywordsCranial Neural Crest ADAM Gene Disintegrin Domain Tropicalis Genome Human ADAM
ADAMs belong to the M12B subfamily of metalloproteinases and metalloproteinase-like proteins . A prototype ADAM is a type I transmembrane protein, but some ADAMs are also present as soluble forms, either due to alternative splicing or protease-mediated cleavage ("shedding") from the cell surface [2, 3]. ADAMs are multi-domain proteins with an extracellular metalloproteinase domain, a disintegrin domain and a cysteine-rich domain; therefore they are also called MDC (metalloproteinase/disintegrin/cysteine-rich) proteins. Some but not all ADAMs contain a canonical zinc-binding motif within the metalloproteinase domain, which is required for protease activity [2, 3]. The disintegrin domain can selectively interact with different integrins ; together with the cysteine-rich domain, it may modulate cell-cell and cell-matrix adhesion [4, 5, 6], as well as substrate recognition by the metalloproteinase domain [7, 8]. The cytoplasmic tail contains binding sites for a variety of cellular proteins, and may be involved in inside-out signaling that regulates the activity of the ectodomain [9, 10, 11].
A phylogenetic tree of ADAMs identified in different species can be found in the tree families database TreeFam . About half of the ADAMs are solely or predominantly expressed in the testis of mammals (i.e. testis-specific), with no orthologue found in nonmammalian species [12, 13]. Functions of these ADAMs are thus mainly related to mammalian reproduction. The other ADAMs are expressed widely in tissues and organs other than the testis. Many of these ADAMs are implicated in embryonic development. Mice lacking or carrying defective ADAM genes often display developmental abnormalities that vary from defects in adipogenesis and myogenesis  and mild dysfuctions in the central nervous system (CNS)[15, 16], to more severe defects such as early embryonic and perinatal lethality [17, 18, 19]. The activities of ADAMs are also linked to a variety of human diseases, such as cancer  and cardiovascular diseases , as well as rheumatoid arthritis and other inflammatory diseases . However, the molecular bases for the functions of ADAMs are largely unappreciated, and identifying the in vivo substrates and binding partners for ADAMs remains a major challenge [2, 23]. Furthermore, functional redundancy between two or more ADAMs seems to be a common phenomenon [24, 25], posing an additional difficulty for understanding the roles of ADAMs in development and diseases.
One area that has drawn increasing interest concerns the roles of ADAMs in cell signaling, mainly via shedding of cell surface proteins by ADAM metalloproteinase activities. Through this process, ADAMs release key extracellular signaling proteins (e.g. growth factors such as EGF and cytokines such as TNF-α) from the cell surface [3, 22]. ADAMs also participate in the generation of key intracellular signals (e.g. Notch and ErbB4 intracellular domain) by performing an obligate ectodomain cleavage event prior to a regulated intramembraneous proteolytic cleavage [26, 27]. Recently, a subgroup of nonproteolytic ADAMs, ADAMs 11, 22 and 23, were shown to function as receptors for the secreted Leucine-rich glioma inactivated (LGI) proteins, which are required for proper synaptic transmission in the brain [28, 29]. Regulation of cell adhesion and migration is another major function of ADAMs. A large body of evidence suggests that ADAM disintegrin domains can interact with integrins, although most ADAMs do not contain the conserved RGD integrin binding sequence [30, 31]. The cysteine-rich domain of ADAM12 can also mediate cell adhesion by binding cell surface syndecans [6, 32]. Furthermore, ADAM metalloproteinase activities are also involved in remodeling extracellular matrix and regulating cell-cell adhesion through cleavage of extracellular matrix components (e.g. type IV collagen and fibronectin) [33, 34, 35] and adhesion molecules (e.g. cadherins) [36, 37, 38, 39, 40].
The amphibian Xenopus laevis has been used for decades as a model system for studying early vertebrate development. The easy availability of large numbers of fertilized eggs, the ability of the embryos to develop and be manipulated in vitro, and the transparent epidermis of the tadpoles provide some practical advantages over mammalian and other models. However, the pseudo-tetraploid genome and long generation time of this species prevented it from being widely used for genetic and genomic research. These difficulties were overcome in large part by the adoption of Xenopus tropicalis, a closely related species with a true diploid genome and a relatively short generation time. The genome sequence of X. tropicalis is now complete, and large collections of EST clones from both X. tropicalis and X. laevis are available, providing useful tools for systematic prediction and characterization of large families of genes. A recently published genome-wide analysis of the matrix metalloproteinases (MMPs), another family of extracellular metalloproteinases that is related to the ADAMs, revealed evolutionary conservation and distinct duplication patterns of MMPs in X. tropicalis as compared to humans and other mammals . Several ADAM genes have been cloned previously from X. laevis and characterized for their functions in fertilization and development [34, 42, 43, 44, 45]. Given the key roles of these ADAMs in vivo, it is important to establish their orthologues in mammals and to identify their paralogues in the Xenopus genome.
As an initial step for systematic studies of all ADAM family proteins in the frog species, we started out to identify the homologues of known vertebrate ADAMs that are expressed in X. tropicalis. Our study shows that most non-testis specific ADAMs are conserved between frogs and mammals. However, some potential loss or gain of ADAM genes (and possibly functions) in X. tropicalis as compared to mammals suggests evolutionary divergence of ADAMs in this amphibian species.
Results and discussion
ADAM genes in the X. tropicalis genome
Searches of the current version (v4.1) of the X. tropicalis genome assembly yielded homologues for most known vertebrate non-testis specific ADAMs. Sequences of these predicted ADAM genes were then used to search against the EST databases (or to design primers for experimental cloning) to obtain full-length cDNA clones from X. tropicalis and, where possible, X. laevis. No orthologues of the mammalian testis-specific ADAMs have been found in any nonmammalian species [12, 13]; similarly, we were unable to identify a Xenopus orthologue for any of these ADAMs. A predicted gene encoding the orthologue of X. laevis ADAM16 (previously known as xMDC16), an ADAM expressed only in testis with a potential role in fertilization , was localized to Scaffold_40 of X. tropicalis genome. No orthologue of ADAM16 was found in any other species examined, including mammals, fish and chicken. Therefore it is likely that ADAM16 function is limited to reproduction of the frogs and related species.
X. tropicalis ADAM genes identified in this study
% identity with closest
% identity with
X. laevis orthologue
43 (with ADAM10)
Tailbud stage head; tadpole; metamorphic tail; adult brain (high level)
Gastrula; neurula; tadpole
53 (with ADAM33)
Gastrula; neurula; tadpole; metamorphic tail; adult spleen
Gastrula; metamorphic limb; adult brain, lung, testis
Neurula; adult brain, testis, oviduct
Gastrula; neurula; tadpole
Tadpole; metamorphic brain/spinal cord; adult brain (high level), testis
Tadpole; adult brain (high level), spleen, kidney, thymus
Neurula; tadpole; adult spleen, bone, kidney
In addition to potential loss of ADAM genes as compared to the mammalian genomes, there is also evidence for additional ADAM genes in the X. tropicalis genome. A homologue of ADAM10, which we named ADAM10-like, was found in Xenopus but absent in most placental mammals. We also identified four homologues of ADAM28 on Scaffold_30 of the X. tropicalis genome assembly. Features of X. tropicalis ADAMs identified in this study are discussed in the following sections in terms of different ADAM clades as indicated in Figure 1A.
ADAMs 12, 13 and 19
We previously identified X. laevis ADAM13 as a proteolytically active ADAM expressed predominantly in cranial neural crest (CNC) cells , an embryonic cell population that gives rise to craniofacial structures in vertebrates [55, 56]. Ectopic expression of a protease-dead mutant of ADAM13, as well as antisense morpholino mediated knockdown of ADAM13, inhibits CNC migration in neurula stage embryos [34, 40]. A closely related ADAM, ADAM19, was recently found to be required for CNC induction and migration in X. laevis . The similarity between the in vivo functions of ADAMs 13 and 19 raises the possibility that these two ADAM metalloproteinases may act through similar mechanisms (e.g. by cleaving the same physiologically relevant substrates). Furthermore, there may be additional paralogues with similar roles in CNC development encoded by the frog genome. To address these questions we performed a search for closely related homologues of X. laevis ADAMs 13 and 19 in X. tropicalis.
Human ADAM15 was originally named metargidin (metalloprotease-RGD-disintegrin protein), because it was the first cellular disintegrin protein (and to date the only human ADAM) found to contain the consensus RGD integrin binding site in the disintegrin domain . However, this RGD sequence is not conserved in mouse ADAM15 . The mammalian ADAM15 gene encodes an active metalloproteinase, and several substrates have been identified for this enzyme [59, 60, 61]. Mice lacking ADAM15 do not show any apparent deficiencies, but they are more resistant to pathological neovascularization .
ADAM28 (also known as MDC-L or eMDC II) is a proteolytically active ADAM that is highly expressed in the epididymis and in lymphocytes [65, 66, 67]. Several alternatively spliced forms of ADAM28 have been detected in vivo, including a soluble form without a transmembrane region or cytoplasimc tail [66, 67]. ADAM7, although proteolytically inactive, is closely related to ADAM28 (Figure 1). Genes encoding ADAM7, ADAM28, and ADAMDEC1 (Decysin) form a metalloproteinase gene cluster on human chromosome 8p12, presumably as a result of gene duplication . ADAMDEC1 is a soluble ADAM-like protein lacking part of the disintegrin domain and the entire cycteine-rich domain; a conserved histidine residue in the zinc-binding motif is replaced by aspartate, but such a replacement was thought to have no negative effect on the metalloproteinase activity . Expression of ADAMDEC1 is restricted to the immune system and is regulated by various stimuli during monocyte differentiation .
As discussed above, no Xenopus orthologue of ADAM7 was identified in this analysis. ADAMDEC1 seems to exist only in mammals , and we were unable to identify any likely orthologue in the X. tropicalis genome or in X. tropicalis/X. laevis EST databases. However, a BLAST search against the X. tropicalis genome assembly yielded four potential genes possibly encoding ADAM28 homologues on Scaffold_30 (Figure 2K). Although these potential genes have only slightly higher sequence similarities to ADAM28 than to ADAM7, the deduced protein sequences all contain a conserved zinc-binding motif (Additional File 3 and data not shown). Therefore they are considered homologues of adam28 (as ADAM7 is proteolytically inactive and seems to be restricted to mammals), and were assigned the names adams 28a-d. We obtained full-length cDNA clones for adams 28a and 28b, and found two partial clones for adam28c (CX934006 and CF377167). In addition, we identified a full-length cDNA encoding a soluble form of ADAM28b (ADAM28bs) in X. laevis EST databases (Additional File 3). The presence of soluble ADAM28 in both humans and frogs indicates a potentially conserved function of a diffusible ADAM28 metalloproteinase in vivo. A sequence alignment for frog and mammalian ADAM28 (including the soluble forms) is shown in Additional File 3. As in the case of human chromosome 8, the cluster of adam28 homologue genes on Scaffold_30 of the X. tropicalis genome may also represent gene duplication events in this region. Sequence comparison of the four X. tropicalis ADAM28 homologues with human ADAM7 and ADAMDEC1 nevertheless indicates no orthologous relationship between them (not shown). Therefore these duplication events in the frog and human genomes may have happened separately after the divergence of amphibians from mammals. Consistent with this hypothesis, both chicken and fish genomes lack such duplication in this region (Figure 2K).
ADAM9 (MDC9) was among the three active ADAM metalloproteinases (the other two were ADAMs 12 and 19) first identified in myoblasts. Because one of these ADAMs, ADAM12, is required for myotube formation in a myoblast cell line and in certain muscle tissues in vivo, these three ADAMs were also called the meltrins [14, 70]. However, ADAM9 is not the most closely related paralogue of ADAMs 12 and 19 (Figure 1), and no apparent phenotype in muscle development was observed in adam9 -/- mice, although a recent report showed that they eventually develop retinal degeneration . These findings suggest that ADAM9 does not belong to the meltrin clade.
ADAMs 11, 22 and 23
Three proteolytically inactive ADAMs, ADAMs 11, 22 and 23 (also known as MDC, MDC2 and MDC3, respectively), form an ADAM clade that is highly expressed in the brain . Studies with knockout mice revealed that each of these ADAMs is required for normal neuronal function [15, 16, 49, 50]. It was recently shown that ADAM22 functions as a receptor for the secreted protein LGI1. Mutations in LGI1 cause heritable epilepsies in humans, and one of these mutated forms of LGI1 fails to bind ADAM22 [29, 72]. In a separate study, ADAMs 11, 22 and 23 were all shown to interact with LGI1 and the closely related LGI4 , suggesting that there may be a common mechanism for these ADAMs in maintaining normal brain function. Two X. laevis cDNA clones encoding different ADAMs, one partial (for xMDC11a) and one full-length (for xMDC11b), were described previously . Our sequence comparison with the X. tropicalis and human genomes indicates that they are orthologues of mammalian ADAMs 11 and 22, respectively (not shown). X. laevis ADAM11 is highly expressed in CNC cells , but no functional study for this ADAM has been reported to date.
ADAMs 10, 10-like and 17
ADAMs 10 and 17 are probably the most extensively studied ADAMs because of their important roles in development and disease. ADAM10 (also known as Kuzbanian or KUZ) was originally discovered in Drosophila as an essential component of the Notch signaling pathway , while ADAM17 (also known as tumor necrosis factor-α converting enzyme or TACE) was first cloned in mice as the major enzyme required for shedding of the cytokine TNF-α [75, 76]. ADAMs 10 and 17 share many substrates, and it is still a subject of debate as to which of these two enzymes is required for cleaving Notch receptors [77, 78]. The Xenopus system has long been used to study Notch signaling, and ectopic expression of a dominant-negative form of mouse ADAM10 was shown to cause a Notch phenotype in X. laevis embryos . Therefore it is important to identify the Xenopus homologues of ADAMs 10 and 17 and understand their respective roles in early development.
ADAMs are widely involved in developmental and pathological processes through their activities as proteases and binding partners for other proteins. The recent emergence of Xenopus tropicalis as a powerful new vertebrate model for developmental and genetic studies prompted us to take a genome-wide approach to identify ADAM genes existing in this species. Using bioinformatics tools, we were able to obtain full-length sequence information for putative X. tropicalis orthologues of most mammalian non-testis specific ADAMs. The orthology between X. tropicalis and human ADAMs were further supported by conserved splicing patterns and synteny. The high sequence similarities between mammalian and Xenopus ADAMs 9, 10, 11, 12, 17, 19, 22 and 23 are in good agreement with their essential roles in vivo, as demonstrated by previous studies with knockout mice. Our sequence analyses also revealed the conservation of certain features, such as alternative splicing and cytoplasmic SH3 binding motifs, in some ADAMs. Functional characterization of these ADAMs (and specific motifs) in X. tropicalis may provide useful information to guide future research on human development and diseases. Evolutionary divergence is also evident by the potential loss of some ADAM genes and ADAM domains/motifs in Xenopus. Orthologues of ADAMs 7 and 8 were not detected in the current version of X. tropicalis genome sequence, and ADAM15 protease activity and part of the ADAM11 propeptide seem to be absent in X. tropicalis. These findings suggest that functions of these ADAMs or ADAM domains are likely not conserved. In contrast, the additional ADAM10- and ADAM28-like metalloproteinases, which presumably arose as results of gene duplication, may have conferred selective advantages during the evolution of amphibians. The sequence information described in this study provides the basis for understanding the evolution of ADAM functions in vertebrates.
Homologues of known human or X. laevis ADAM genes were identified by using sequence-based searches of the Joint Genome Institute (JGI) X. tropicalis genome assembly v4.1 , and cDNA constructs were obtained from IMAGE Consortium distributor (OpenBiosystems) or by experimental cloning as described below. All new X. tropicalis ADAM cDNAs reported here have been sequenced by the Biomolecular Research Facility at the University of Virginia, and assembled to give the final full-length sequences. Multiple sequence alignments were constructed with ClustalX 2.0 . Phylogenetic trees were generated using ClustalX (neighbor joining), Phylip neighbor, Phylip protpars (maximum parsimony) and Phylip proml (maximum likelyhood) . Trees were displayed using the Phylip application Drawtree. Because phylogenetic trees generated using different methods are consistent with each other, only those generated with ClustalX are shown in the main figures (see Additional File 6 for trees generated using the other models). For bootstrap analyses, bootstrap replicate groups were created using Phylip application Seqboot, and bootstrap values were calculated using Phylip Neighbor, Protpars, and Proml analyses (bootstrap values calculated with ClustalX are very similar to those calculated with Phylip Neighbor, therefore only the latter are shown). Synteny analyses were carried out using Metazome . Exon-intron structures were estimated by comparing the cDNA sequences with X. tropicalis genome sequence using BLAT searching from the UCSC Genome Bioinformatics Site . Signal peptidase cleavage sites were assigned according to available sequence annotations (for known ADAMs) or predictions by SignalP (for new ADAMs identified in this study) . Domain structure predictions were carried out using the Simple Modular Architecture Research Tool (SMART) .
The following human ADAM protein sequences were used to identify their X. tropicalis homologues: ADAM9 (Q13443), ADAM10 (O14672), ADAM11 (O75078), ADAM12 (O43184), ADAM15 (Q13444), ADAM17 (P78536), ADAM19 (Q9H013), ADAM22 (Q9P0K1), ADAM23 (O75077), ADAM28 (Q9UKQ2), and ADAM33 (Q9BZ11). Previously reported X. laevis ADAMs used in this study are: ADAM9/xMDC9 (NP_001079073), ADAM10 (Q8JIY1), ADAM11/xMDC11a (Q9PSZ3), ADAM13 (AAI69959), ADAM19 (ACE82293), and ADAM22/xMDC11b (O42596). IMAGE clone IDs for ESTs encoding X. tropicalis and X. laevis ADAMs identified in this study are: X. tropicalis ADAM9 (7644280), ADAM10 (7644709), ADAM11 (7650432), ADAM15 (7693815), ADAM17 (6994025), ADAM22 isoform 1 (7658598), ADAM22 isoform 2 (8916700), ADAM22 isoform 3 (7650959), ADAM23 (7649052), ADAM28a (7716115), and ADAM28b (8898681); X. laevis ADAM15 (7978429; GenBank accession # BC146626), ADAM17 (3199617; GenBank accession # DQ287907), and ADAM28bs (5513111; GenBank accession #BC091726).
Cloning and RT-PCR
Total RNA from X. tropicalis embryos at desired stages was prepared as described . X. tropicalis orthologues of X. laevis adam13 and mammalian adams 12 and 19, and X. tropicalis adam10-like gene were identified by searching against the X. tropicalis genome assembly, and the predicted sequences were used to design primers for RT-PCR to obtain partial clones. 5'- and 3'- RACE was then performed using BD Smart RACE kit (BD Biosciences), and full-length cDNA sequences were assembled into pCR2.1. RT-PCR experiments for expression of adams 12, 13, and 19 were carried out using total RNA purified from X. tropicalis embryos at different developmental stages. Primer sequences are shown in Additional File 7.
This work was supported by the National Institute of Health (DE14365 and HD26402 to DWD) and March of Dimes Foundation (grant F405-140 to JMW and 1-FY10-399 to DWD and SW). SW was supported by an American Heart Association postdoctoral fellowship, and LCB was supported by a training grant to the University of Virginia (5T32GA09109) and by a Ruth L. Kirschstein postdoctoral fellowship.
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