Link Protein Has Greater Affinity for Versican than Aggrecan

Shuiliang Shi,Suzanne Grothe,Yiping Zhang,Maureen D O'Connor-Mccourt,A Robin Poole,Peter J Roughley,John S Mort

doi:10.1074/jbc.m310091200

Abstract

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.

Highlights

Sists of one or two EGF repeats, a C-type lectin-like motif, and a complement regulatory protein-like repeat [3]
The G1 domains of aggrecan (AG1) and versican (VG1) and Link protein (LP) were expressed as His-tagged recombinant proteins with their own signal peptide
N-terminal sequencing analysis showed that recombinant versican G1 (VG1) has a single N-terminal sequence (LHKVKVGKSPP—-) identical to that reported for the mature core protein, that the recombinant aggrecan G1 (AG1) preparation contained two N-terminal sequences: VTVETSDHDNSLSV— and VETSDHDNSLSV, representing the mature core protein and cleavage two residues downstream

Summary

The abbreviations used are

G, globular; AG1, aggrecan G1 globular domain; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay; HA, hyaluronan; LP, link protein; PBS, phosphate-buffered saline; RU, response unit; SPR, surface plasmon resonance; VG1, versican G1 globular domain

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION