Abstract
Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.
Highlights
Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues
The Different Proteoglycan Lectin Domain Ligands Compete for the Same Binding Site—In solid phase assays using a surface coated with fibulin-2, we allowed alkaline phosphatasetagged aggrecan or versican rCLD to bind to the coated protein in the presence of varying concentrations of the different ligands
In this work we demonstrate that fibulin-2 is a high affinity ligand for the C-type lectin domains of the proteoglycans aggrecan, brevican, and versican
Summary
Production of Recombinant Alkaline Phosphatase-tagged Lectin Domains—The construction of His-tagged rCLD mammalian expression pCEP4 plasmids have previously been described [15, 18]. After washing the wells with TTBS-Ca or TTBS-EDTA, radiolabeled and affinity-purified aggrecan or versican at different concentrations was added at a starting dilution of 100,000 and 40,000 cpm/well, respectively, in triplicates in either TTBS-Ca or TTBS-EDTA. For rotary shadowing 20-l aggrecan samples (typical concentrations 10 –20 g/ml) were dialyzed overnight at 4 °C against 0.2 M ammonium hydrogen carbonate, pH 7.9 They were subsequently mixed with equal volumes of 80% glycerol and sprayed onto freshly cleaved mica with a nebulizer designed for small volumes. For negative staining 5-l samples of complexes between fibulins and native aggrecan or fibulins and rCLDs (typical concentrations 5–10 g/ml in TBS/5 mM CaCl2) were adsorbed to 400mesh carbon-coated copper grids, washed briefly with water, and stained with 0.75% uranyl formate. An antiserum was raised against recombinant rat aggrecan lectin domain and will be described in detail elsewhere
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