Structural and functional studies on hyaluronan‐protein aggregates produced in vitro
Authors: Seyfried Nicholas T. , McVey Gillian F. , Dudhia Jayesh , Day Anthony J. 
Published: August 2004

Introduction  The proteoglycan aggrecan forms link protein‐stabilized complexes with hyaluronan (HA) that provide cartilage with its load bearing properties. Aggrecan binding to both HA and cartilage link protein are mediated through its N‐terminal G1‐domain, which is composed of an immunoglobulin fold and two contiguous Link modules. Similar aggregates (potentially containing members of the new link protein family) in which the proteoglycan versican substitutes for aggrecan are likely to contribute to the structural integrity of many other tissues.

Materials and methods  Human cartilage link protein (LP1) and the G1‐domains of human aggrecan and versican were cloned and expressed in Drosophila S2 cells. The recombinant proteins were purified from media using a combination of ion‐exchange chromatography and reverse‐phase HPLC. A microtitre plate assay was used to determine the minimum length of HA necessary for high affinity binding; the interaction of biotinylatd‐LP1 to immobilized G1‐domains was also determined. The ability of the recombinant proteins to form complexes in solution was assessed with HA oligosaccharides of defined length (i.e. HA10‐HA40) using a combination of gel filtration and protein cross‐linking experiments. Cross‐linking products were identified by in‐gel digestion with trypsin followed by MALDI‐TOF mass spectrometry.

Results  SDS‐PAGE showed the three recombinant proteins to be highly pure and that they all contained N‐linked glycosylation; amino acid sequencing revealed that aggrecan has undergone some differential processing at its N‐terminus. LP1, G1‐aggrecan and G1‐versican were all functionally active showing HA‐binding properties that are similar to those described for the native proteins; e.g. HA10 was the minimum length that could compete effectively for polymeric HA binding. Gel filtration and protein cross‐linking studies reveal that LP1 and G1‐aggrecan interact in both the absence and presence of HA. Interestingly, LP1 and G1‐versican did not interact in the absence of HA yet could be cross‐linked in the presence of HA24 but not HA20 or shorter oligosaccharides. However, when G1‐versican was immobilized on a microtitre plate, it was found to interact with biotinylated‐LP1, which is consistent with recent reports in the literature. Treatment of G1‐aggrecan or G1‐versican with PNGase F (which removes most of the N‐linked glycans from these proteins) did not affect the formation of aggregates in the presence of HA, indicating that glycosylation is not required for structural integrity or functional activity. Surprisingly, for G1‐aggrecan HA of approximately 40 sugar units appears to be the minimum size that can fully accommodate two protein molecules, whereas in the case of versican this occurs with HA between HA20 and HA24.

Conclusion  Aggrecan and versican appear to have distinct properties both with regard to their interaction with LP1 and HA, consistent with the hypothesis that different hyaladherins bind and capture distinct conformations of HA (Day and Sheehan 2001). Work is now in progress to produce aggrecan/LP1 and versican/LP1 ternary complexes for crystal studies.