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.

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 chondr...

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Main Authors: Seyfried, N, McVey, G, Almond, A, Mahoney, D, Dudhia, J, Day, A
Format: Journal article
Language:English
Published: 2005
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author Seyfried, N
McVey, G
Almond, A
Mahoney, D
Dudhia, J
Day, A
author_facet Seyfried, N
McVey, G
Almond, A
Mahoney, D
Dudhia, J
Day, A
author_sort Seyfried, N
collection OXFORD
description 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.
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spelling oxford-uuid:5a290153-a795-41c2-9b69-03286be508062022-03-26T17:14:07ZExpression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:5a290153-a795-41c2-9b69-03286be50806EnglishSymplectic Elements at Oxford2005Seyfried, NMcVey, GAlmond, AMahoney, DDudhia, JDay, AThe 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.
spellingShingle Seyfried, N
McVey, G
Almond, A
Mahoney, D
Dudhia, J
Day, A
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.
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.
title_full 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.
title_fullStr 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.
title_full_unstemmed 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.
title_short 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.
title_sort 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
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