title: Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C and other extracellular matrix proteins.
abstract: 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.
authors.affiliation: The Wellcome Trust Centre for Cell Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Day
authors.forename: Anders I
authors.initials: AI
authors.lastname: Olin
authors.forename: Alan D
authors.initials: AD
authors.lastname: Murdoch
authors.forename: Ann
authors.initials: A
authors.lastname: Canfield
authors.forename: Takako
authors.initials: T
authors.lastname: Sasaki
authors.forename: Rupert
authors.initials: R
authors.lastname: Timpl
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Hardingham
authors.forename: Anders
authors.initials: A
authors.lastname: Aspberg
mesh_terms: D053674:Aggrecans; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D019902:Chondrocytes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D037102:Lectins; D037181:Lectins, C-Type; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000096:biosynthesis / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D019063:Tenascin / Q000737:chemistry*; D013997:Time Factors
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D037102:Lectins; D037181:Lectins, C-Type; D008024:Ligands; D011509:Proteoglycans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019063:Tenascin; D004815:Epidermal Growth Factor