Alternative Splicing in the Aggrecan G3 Domain Influences Binding Interactions with Tenascin-C and Other Extracellular Matrix Proteins

• Published in: The Journal of biological chemistry Vol. 279; no. 13; pp. 12511 - 12518
• Main Authors: Day, Joanna M., Olin, Anders I., Murdoch, Alan D., Canfield, Ann, Sasaki, Takako, Timpl, Rupert, Hardingham, Timothy E., Aspberg, Anders
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.03.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Aggrecans; Alternative Splicing; Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Basic Medicine; Binding Sites; Binding, Competitive; Cell and Molecular Biology; Cell- och molekylärbiologi; Chondrocytes - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor - chemistry; Extracellular Matrix - metabolism; Extracellular Matrix Proteins; Fibronectins - chemistry; Humans; Kinetics; Lectins; Lectins, C-Type; Ligands; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Proteoglycans - biosynthesis; Proteoglycans - chemistry; Recombinant Proteins - chemistry; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Surface Plasmon Resonance; Tenascin - chemistry; Time Factors
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M400242200

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectin-binding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.