Abstract
Thrombospondins (THBSs) are a family of secreted calcium-binding glycoproteins with roles in angiogenesis, cell motility, apoptosis, cytoskeletal organization, and extracellular matrix organization. The THBS-2 signature domain (three epidermal growth factor (EGF)-like modules, a wire module with 13 calcium-binding repeats, and a lectin-like module) binds 30 calcium ions and forms extensive interactions among its parts. We explored the significance of these structural elements by examining the impact of 10 different mutations known to result in pseudoachondrodysplasia or multiple epiphyseal dysplasia when found in the homologous wire and lectin-like modules of thrombospondin-5 (THBS-5). A variety of observations indicate that the mutations result in unstable THBS-5 proteins that aggregate in the endoplasmic reticulum. We introduced the mutations into homologous sites of a THBS-2 construct, for which the crystal structure is known, and determined the effects of the mutations on structure as assayed by differential scanning calorimetry and expression of the epitope for the 4B6.13 conformation-sensitive antibody. Abnormalities were found in one or more of several readouts: stability of interactions between the wire and lectin-like modules, stabilities of the EGF-like and wire modules, expression of the 4B6.13 epitope in soluble protein, and expression of the 4B6.13 epitope in substrate-adsorbed protein at different calcium concentrations. The patterns of abnormalities support the idea that the EGF-like, wire, and lectin-like modules constitute a dynamic and interactive calcium-sensitive structure in which a distortion at one site is transmitted to distal sites, leading to global changes in the protein.
Highlights
The competition ELISA column shows values for binding versus protein concentrations and in parentheses, concentration of calcium required for 50% binding to surface-adsorbed full-length THBS-2, using 1 M protein
The direct ELISA column shows values for binding versus calcium concentrations and in parentheses, concentration of protein during adsorption required for half maximal epitope expression in 2 mM calcium
Disruption of Protein Stability Is Specific for Disease-associated Mutations—As a control for the changes found in the DSC profile, we studied the signature domain of wild-type mouse THBS-2, which has the same spacing of residues as the human protein and changes in residues at 37 sites, and mouse THBS-2 containing N703L and K722H mutations in repeat 1C
Summary
Protein Cloning—The pAcGP67.coco baculovirus transfer vector encoding a secretion signal peptide 5Ј to the cloning site followed by DNA encoding a short linker and six-histidine tag, was used to enable baculovirus-driven protein expression, secretion, and subsequent purification [27]. Enzyme-linked Immunosorbent Assay (ELISA)—Proteins in 2 mM calcium were tested in competition ELISA for binding to 4B6.13 as described [26] to assess whether various concentrations of soluble protein in 2 mM calcium were able to compete with surface adsorbed recombinant full-length THBS-2 for binding of the monoclonal antibody These competition ELISA experiments were repeated, each with triplicate data points, on multiple occasions and with at least two separate preparations of protein. Polyclonal rabbit anti-human THBS-2 antibodies [29] were used to test whether the various proteins were adsorbed to the plate at similar concentrations These direct ELISA experiments were repeated, each with triplicate data points, on multiple occasions and with at least two separate preparations of protein.
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