title: Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.
abstract: The chondroitin sulfate proteoglycan aggrecan forms link protein-stabilized complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other chondroitin sulfate proteoglycans (i.e. versican, brevican, and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this study, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells. The recombinant human proteins were found to have properties similar to those described for the native molecules (e.g. cLP was able to form oligomers, and HA decasaccharides were the minimum size that could compete effectively for their binding to polymeric HA). Gel filtration and protein cross-linking/matrix-assisted laser desorption ionization time-of-flight peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other in solution yet formed ternary complexes with HA24. N-linked glycosylation of AG1 and VG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Surprisingly, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences in the conformation of HA stabilized on binding these proteins.
authors.affiliation: Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.forename: Gillian F
authors.initials: GF
authors.lastname: McVey
authors.forename: Andrew
authors.initials: A
authors.lastname: Almond
authors.forename: David J
authors.initials: DJ
authors.lastname: Mahoney
authors.forename: Jayesh
authors.initials: J
authors.lastname: Dudhia
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D037181:Lectins, C-Type; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D053675:Versicans; D000099249:Proteoglycan Link Protein
chemical_list: D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid; D037181:Lectins, C-Type; D010446:Peptide Fragments; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011509:Proteoglycans; D011994:Recombinant Proteins; D012996:Solutions; D053675:Versicans; D000099249:Proteoglycan Link Protein; C508322:VCAN protein, human