Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex

Abstract

The evolution of cadherins, which are essential for metazoan multicellularity and restricted to metazoans and their closest relatives, has special relevance for understanding metazoan origins. To reconstruct the ancestry and evolution of cadherin gene families, we analyzed the genomes of the choanoflagellate Salpingoeca rosetta, the unicellular outgroup of choanoflagellates and metazoans Capsaspora owczarzaki, and a draft genome assembly from the homoscleromorph sponge Oscarella carmela. Our finding of a cadherin gene in C. owczarzaki reveals that cadherins predate the divergence of the C. owczarzaki, choanoflagellate, and metazoan lineages. Data from these analyses also suggest that the last common ancestor of metazoans and choanoflagellates contained representatives of at least three cadherin families, lefftyrin, coherin, and hedgling. Additionally, we find that an O. carmela classical cadherin has predicted structural features that, in bilaterian classical cadherins, facilitate binding to the cytoplasmic protein β-catenin and, thereby, promote cadherin-mediated cell adhesion. In contrast with premetazoan cadherin families (i.e., those conserved between choanoflagellates and metazoans), the later appearance of classical cadherins coincides with metazoan origins.

Fig. 1.

Phylogenetic distribution and abundance of cadherins in the genomes of diverse eukaryotes. Once thought to be restricted to metazoans, cadherins are abundant in choanoflagellates and evolved before the divergence of Capsaspora owczarzaki, choanoflagellates, and metazoans (1). EC domains detected in the genome of the oomycte Pythium ultimum likely evolved through convergence or lateral gene transfer (9). The number of cadherin families inferred at ancestral nodes (determined based upon their shared domain composition and organization) is indicated (open circles). The dashed lineage of Trichoplax adhaerens reflects its uncertain phylogenetic placement. *All fungal and plant species represented in the Pfam v24.0 database (29) were analyzed. Aque, A. queenslandica; Cele, Caenorhabditis elegans; Cint, Ciona intestinalis; Cowc, C. owczarzaki; Ddis, Dictyostelium discoideum; Dmel, D. melanogaster; Hmag, Hydra magnipapillata; Mbre, M. brevicollis; Mmus, Mus musculus; Nvec, N. vectensis; Pult, P. ultimum; Sros, S. rosetta; Tadh, T. adhaerens.

Fig. 2.

Predicted domain architecture of modern representatives of premetazoan cadherins. At least three cadherin families evolved before the origin of metazoans. (A) The single cadherin discovered in the genome of C. owczarzaki has a cassette of EGF repeats positioned proximal to a single transmembrane domain (blue box) that is also found in choanoflagellate and sponge cadherins. The phylogenetic relationships among cadherins with this feature are not yet clear. The lefftyrin (B) and coherin (C) families are present only in choanoflagellates and sponges. Lefftyrins are distinguished by an N-terminal “LEF” cassette (orange box) with a Lam-N domain, four EGF repeats, and a Furin repeat and a C-terminal “FTY” cassette (purple box) with one or two Fibronectin 3 domains, a transmembrane domain, and a tyrosine phosphatase domain. Coherins contain a diagnostic bacterial/archaeal-like cohesin (50) domain. (D) The hedgling family (1, 26) is present in choanoflagellates, sponges and cnidarians and is absent from bilaterians. All hedglings contain an N-terminal Hedgehog signal domain linked to a von Willebrand A domain (green box) and most contain a series of EGF repeats proximal to the transmembrane domain (blue box). Candida ALS, Candida Agglutinin-like sequence; IG I-set, Ig I-set; KU, BPTI/Kunitz family of serine protease inhibitors; Lam-G, Laminin G domain; 9-cystein GPCR, 9-cystein G protein coupled receptor; PKD, polycystic kidney disease; SH2, src homogy domain 2; TNFR, tumor necrosis factor receptor.

Fig. 3.

A conserved β-catenin/classical cadherin protein complex in a sponge. (A) The genome of the sponge O. carmela encodes a classical cadherin, Oc_cdh1, identified by the presence of the diagnostic cadherin cytoplasmic domain (CCD). Oc_cdh1 also has EGF and Lam-G domains in a membrane-proximal position that is typical of invertebrate classical cadherins (4). The dashed line at the N terminus of Oc_cdh1 indicates that the gene model is incomplete because of the draft nature of the genome assembly. (B) An alignment of a portion of the Oc_cdh1 CCD with bilaterian CCDs demonstrates the conservation of two residues (Aspartate and Glutamate, highlighted in green) required for binding to β-catenin (SI Appendix, Fig. S4 depicts the full alignment and includes the only known CCD from the demosponge A. queenslandica, in which critical β-catenin binding residues are also conserved). Conserved residues are shaded gray and Casein Kinase II and Glycogen Synthase Kinase 3b phosphorylation sites essential for the regulation of adhesion dynamics are indicated by filled or open circles, respectively (35, 38, 39). (C) The O. carmela genome also encodes a single β-catenin ortholog (Oc_bcat) with 11 predicted armadillo (arm) repeats and a helix-C domain; each arm repeat is numbered according to its similarity (determined by best-reciprocal Blast) with the 12 arm repeats from other metazoan β-catenin homologs (SI Appendix, Fig. S4). (D) Through comparison of a surface representation of the 3D structure of zebrafish β-catenin (37) with a structural model of Oc_bcat, we predict the conservation of a positively charged groove lined by the third helix (blue) of each arm repeat. Within this groove there are two lysine residues whose orientation resembles that of conserved lysines from zebrafish β-catenin. (E) These lysines align with Lysine-312 and Lysine-435 of mouse β-catenin, each of which are required for binding to mouse E-cadherin (35, 38, 39) at Aspartate-647 and Glutamate-682 (highlighted in B). Ocar_cdh1 was initially discovered from a yeast two-hybrid screen using full-length Ocar_bcat as bait (SI Appendix, Table S2; see SI Appendix for further discussion). CCD, cadherin cytoplasmic domain; EC, extracellular cadherin; EGF, epidermal growth factor domain; Lam-G, Laminin G domain; TM, transmembrane domain.

Fig. 4.

An emerging model of cadherin evolution. (A) At least five modern families of cadherins—hedglings, coherins, lefftyrins, CELSR/flamingo and classical cadherins—evolved before the diversification of modern metazoans. Of these families, only the CELSR/flamingo and classical cadherin families are clearly conserved in all metazoan lineages (2, 4, 31). In contrast, among metazoans, hedgling is restricted to sponges and cnidarians. All of the cadherin families that evolved before the divergence of choanoflagellates and metazoans (“premetazoan” cadherin families) have been lost or have evolved beyond recognition in bilaterians. The relationships among the single cadherin detected in the genome of C. owczarzaki (Cowc_Cdh1) and other modern cadherin families are uncertain (indicated by dotted circle, also see Fig. 2A). (B) In addition to having EC domains, members of many cadherin families contain domains that provide clues to their evolutionary origins and to their relationships with other modern protein families (see Discussion).