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

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 disulfidebonded 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. The extracellular matrix (ECM)3 is a complex and dynamic threedimensional environment that plays fundamental roles in morphogenesis and development, tissue structure, repair, and metastasis (1). The tissue-specific expression of collagen types and specialized ECM components results in the formation of architecturally precise interacting networks with unique functional and biological characteristics. A diverse range of ECM components have been described including more than 20 distinct collagen subtypes and a large number of proteoglycans and noncollagenous proteins. Many of these matrix proteins are modular in structure in that they are composed of protein domains, which is important in generating the multifunctionality that is characteristic of ECM proteins (2, 3). One of these domains, found in a growing number of ECM proteins involved in supramolecular structures, is the A domain first described in von Willebrand factor (VWA domain). VWA domains are found in a diverse range of ECM proteins including collagens (types VI, VII, XII, XIV, XX, XXI, XXVII, and XXVIII), matrilins, cochlin, polydom, AMACO (VWA-like domains related to those in matrilins and collagens), and the extracellular portions of nine transmembrane ␣-integrin chains (4 -6)We recently identified a new member of the von Willebrand factor A domain superfamily, WARP (von Willebrand factor A domain-related protein), that may have evolved from a collagen-like molecule (7,8). The WARP protein comprises a single N-terminal VWA domain containing a putative metal ion-dependent adhesion site motif, two fibronectin type III repeats, and a unique C-terminal segment. Our studies demonstrated WARP expression by chondrocytes, and in transfected cells WARP is a secreted glycoprotein that ...

Mice Lacking the Extracellular Matrix Protein WARP Develop Normally but Have Compromised Peripheral Nerve Structure and Function

“…Like many other VWA domain-containing extracellular molecules, WARP was predicted to participate in protein-protein interactions and in the formation of supramolecular structures. Recently WARP has been shown to interact with the heparan sulfate proteoglycan perlecan (3), and in the present study we identify type VI collagen as a ligand for WARP. WARP has a restricted distribution in developing cartilage tissues, where it is expressed at sites of joint cavitation and articular cartilage formation rather than cartilage structures that will undergo endochondral ossification (3).…”

“…Recently WARP has been shown to interact with the heparan sulfate proteoglycan perlecan (3), and in the present study we identify type VI collagen as a ligand for WARP. WARP has a restricted distribution in developing cartilage tissues, where it is expressed at sites of joint cavitation and articular cartilage formation rather than cartilage structures that will undergo endochondral ossification (3). In adult tissues, WARP is highly restricted to the chondrocyte pericellular matrix in articular cartilage and fibrocartilages, where it colocalizes with perlecan and collagen VI (3).…”

“…WARP was immunolabeled with a rabbit anti-C-terminal domain polyclonal antibody or with a sheep polyclonal antibody raised to full-length recombinant WARP (3,5). Perlecan was detected with rat anti-perlecan monoclonal antibody (A7L6, Abcam).…”

“…Our data demonstrate that although WARP is not essential for basement membrane formation or musculoskeletal development, it has critical roles in the structure and function of peripheral nerves. (2,3). Like many other VWA domain-containing extracellular molecules, WARP was predicted to participate in protein-protein interactions and in the formation of supramolecular structures.…”

Three Novel Collagen VI Chains, α4(VI), α5(VI), and α6(VI)

“…This purified eluted antigen was then used to raise antisera in rabbits (Rockland Immunochemicals, Inc.). The resulting antisera were further affinity-purified against the recombinant antigen immobilized on a nitrocellulose membrane as described (24,25). Serum from the same rabbit, taken prior to immunization with the ␣6(VI) antigen, was used as a negative control.…”