WARP Is a Novel Multimeric Component of the Chondrocyte Pericellular Matrix That Interacts with Perlecan

Justin M Allen,John F Bateman,Uwe Hansen,Richard Wilson,Peter Bruckner,Rick T Owens,Takako Sasaki,Rupert Timpl,Jamie Fitzgerald

doi:10.1074/jbc.m513746200

Abstract

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with K(D) values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of "permanent" cartilage structures during development and in mature cartilages.

Highlights

Ular in structure in that they are composed of protein domains, which is important in generating the multifunctionality that is characteristic of extracellular matrix (ECM) proteins [2, 3]
Found in a growing number of ECM proteins involved in supramolecular structures, is the A domain first described in von Willebrand factor (VWA domain)
We show that WARP can strongly interact with perlecan, providing a mechanism by which WARP is integrated into the extracellular matrix of cartilage

Summary

EXPERIMENTAL PROCEDURES

CDNA Constructs and Cell Culture—The hexahistidine-tagged WARP constructs in the pCEP4 vector [7] containing single Cys-to-Ser mutations at amino acid 369 or 393 and the double mutant (C369S/ C393S) were generated by strand overlap extension PCR [9]. The wildtype and mutant His-WARP cDNAs were transfected into HEK 293EBNA cells using FuGENE 6 transfection reagent (Roche Applied Science) and grown as described [7]. WARP Immunoprecipitations—Transfected cells were grown to confluence in 35-mm dishes and labeled for 18 h with 300 ␮Ci of L-[35S]methionine (1388 Ci/mmol; PerkinElmer Life Sciences) in Dulbecco’s modified Eagle’s medium as described previously [7]. The cell and medium fractions were collected in the presence of protease inhibitors, and His-tagged WARP was immunoprecipitated from the cell and medium fractions with anti-His antibody (Roche Applied Science) (0.5 ␮g/ml) and protein A-Sepharose overnight. The immunoprecipitated material was denatured and fractionated on a 7.5% (v/v) SDS-polyacrylamide gel and subjected to fluorography

WARP Interacts with Perlecan

RESULTS

DISCUSSION