The evolution of thrombospondins and their ligand-binding activities

Abstract

The extracellular matrix (ECM) is a complex, multiprotein network that has essential roles in tissue integrity and intercellular signaling in the metazoa. Thrombospondins (TSPs) are extracellular, calcium-binding glycoproteins that have biologically important roles in mammals in angiogenesis, vascular biology, connective tissues, immune response, and synaptogenesis. The evolution of these complex functional properties is poorly understood. We report here on the evolution of TSPs and their ligand-binding capacities, from comparative genomics of species representing the major phyla of metazoa and experimental analyses of the oligomerization properties of noncanonical TSPs of basal deuterostomes. Monomeric, dimeric, trimeric, and pentameric TSPs have arisen through separate evolutionary events involving gain, loss, or modification of a coiled-coil domain or distinct domains at the amino-terminus. The relative transience of monomeric forms under evolution implicates a biological importance for multivalency of the C-terminal region of TSPs. Most protostomes have a single TSP gene encoding a pentameric TSP. The pentameric form is also present in deuterostomes, and gene duplications at the origin of deuterostomes and gene loss and further gene duplication events in the vertebrate lineage gave rise to distinct forms and novel domain architectures. Parallel analysis of the major ligands of mammalian TSPs revealed that many binding activities are neofunctions representing either coevolutionary innovations in the deuterostome lineage or neofunctions of ancient molecules such as CD36. Contrasting widely conserved capacities include binding to heparan glycosaminoglycans, fibrillar collagen, or RGD-dependent integrins. These findings identify TSPs as fundamental components of the extracellular interaction systems of metazoa and thus impact understanding of the evolution of ECM networks. The widely conserved activities of TSPs in binding to ECM components or PS2 clade integrins will be relevant to use of TSPs in synthetic extracellular matrices or tissue engineering. In contrast, the neofunctions of vertebrate TSPs likely include interactions suitable for therapeutic targeting without general disruption of ECM.

FIG. 1.

Evolution of thrombospondins in the metazoa. Accession numbers are from GenBank (+), JGI (*) or HGSC (^). In the domain models, red lines indicate a coiled coil; short vertical lines indicate positions of paired cysteine residues; dotted lines incomplete sequence regions. coil, coiled coil; CX2X, CX₂C domain; EGF, EGF domain; LG, laminin G–like domain; L-lectin, L-type lectin-like domain; vWF_C, von Willebrand factor type C domain.

FIG. 2.

Unrooted phylogenetic tree of the TSPs. Sequences of 450 residues including the type 3 repeats and L-lectin–like domain from 33 TSPs representing basal metazoa, protostomes, and the deuterostome TSP family were aligned in MUSCLE and analyzed in PHYML with 100 bootstrap cycles. Bootstrap values are given for major internal nodes. Branch length is proportional to the number of substitutions/site. The three subgroups of deuterostome TSPs are well supported, and the basal and protostome TSPs exhibit high sequence diversity with some individual sequences appearing most closely related to the A or DD subgroups. Bf, Branchiostoma floridae; Cc, Capitella capitella; Ci, Ciona intestinalis; Dp, Daphnia pulex; Dm, Drosophila melanogaster; Hm, Hydra magnipapillata; Hs, Homo sapiens; Lg, Lottia gigantea; Mj, Marsupenaeous japonicus; Nv, Nematostella vectensis; Sk, Saccoglossus kowalevskii; Sp, Strongylocentrotus purpuratus; Ta; Trichoplax adhaerens; Tc, Tribolium castena; Tr, Takifugu rubripes.

FIG. 3.

Oligomerization properties of Ciona TSP-A and TSP-DD. (A) Oligomerization of Ciona intestinalis TSP-A. TSP-A and Drosophila TSP were collected from conditioned media of transiently transfected COS-7 cells onto heparin–Sepharose, resolved on 4–10% polyacrylamide gradient gels under reducing or nonreducing conditions, and analyzed by immunoblotting. Migration positions of purified ECM glycoproteins used nonreduced as additional markers are shown to the left of the panel. (B) The IVR has independent dimerization activity. The 6His-tagged IVR of TSP-A was inducibly expressed in Escherichia coli, purified on metal affinity beads, resolved on 15% polyacrylamide gels under reducing or nonreducing conditions, and analyzed by immunoblotting. (C,D) The DD and CX₂C domain of C. intestinalis TSP-DD lack oligomerization activity. Tagged versions of the discoidin domain (C) or CX2C domain (D) were collected from conditioned media of transiently transfected COS-7 cells (C) or bacterial lysates (D) onto metal affinity beads, resolved on 12.5% polyacrylamide gels under reducing or nonreducing conditions, and analyzed by immunoblotting.

FIG. 4.

Representation of TSP ligand-binding motifs and their cognate ligands across the metazoa. Results from Hydra and Drosophila are representative of other basal metazoa and protostomes. White boxes, ligand not encoded within the indicated species genome; diagonal line, ligand encoded; black box, the ligand and a TSP with the appropriate binding motif are both encoded.

FIG. 5.

Model of TSP evolution in the metazoa. See text for details. Details of TSP family evolution in vertebrates can be found in McKenzie et al. 2006).