Introduction PG-M/versican is a chondroitin sulfate proteoglycan located in the extracellular matrix (ECM). It contains an N-terminal globular domain (G1) and a C-terminal globular domain (G3), also known as the selectin-like domain, with a large central domain containing sites for glycosaminoglycan modification (Ito et al., 1995; Kiani et al., 2002). The G3 domain contains two epidermal growth factor (EGF)-like sequences, a carbohydrate recognition domain (CRD) and a complement binding protein (CBP)-like domain. This proteoglycan is expressed by a large variety of tissues (Wight, 2002). It is important in heart development (Henderson and Copp, 1998): disruption of the versican gene results in the abnormal development of the heart, and this is lethal (Mjaatvedt et al., 1998). Versican is also highly expressed by aortic endothelial cells and vascular smooth muscle cells (Yao et al., 1994). In vitro studies have shown that versican modulates arterial and vascular smooth muscle cell adhesion, migration and proliferation (Lemire et al., 2002). As a matrix molecule, versican is known to associate with a number of molecules in the ECM and on the cell surface (Wight, 2002; Wu et al., 2004). We have demonstrated that versican enhances cell proliferation and differentiation (Zhang et al., 1998a; Zhang et al., 1998b). The EGF-like motifs in the G3 domain of versican are involved in this effect (Wu et al., 2001), and we hypothesized that versican plays this role through binding to cell surface proteins. We used a yeast two-hybrid approach with the G3 domain as bait to screen a human cDNA library, and thus identified P- selectin glycoprotein ligand-1 (or PSGL-1) as the versican binding protein. PSGL-1 is a homodimeric glycoprotein held together by disulfide-bonds expressed on the cell surface of leukocytes, which mediates leukocyte rolling on the vascular endothelium (Pouyani and Seed, 1995; Sako et al., 1993). Leukocyte locomotion from the blood stream into tissues in response to inflammatory stimuli is a critical component of the immune response, which involves the activities of a range of adhesion and signaling molecules. The first events in this process are leukocyte adhesion and leukocyte rolling on the endothelial surface under vascular shear flow, mediated by selectins. PSGL-1 is expressed by essentially all blood leukocytes including lymphocytes, monocytes, neutrophils and platelets, and has been shown to mediate the rolling of human neutrophils on selectins (Moore et al., 1995). The N-terminal fragment of PSGL-1 is extensively glycosylated, which is critical for binding to the selectins (Leppanen et al., 2002). Here we report that PSGL-1 also binds to versican G3 domain via a motif different from the selectin-binding domain. The binding induces human leukocyte aggregation, and this finding suggests a novel role for PSGL-1 in this activity. Materials and Methods Materials Lipofectin, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin/EDTA, restriction endonucleases and T4 DNA ligase were purchased from Invitrogen. ECL western blot detection kit was from Amersham. Horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG was from Sigma. Anti-His-tag monoclonal antibody and DNA Midi-prep kit were purchased from 5887 P-selectin glycoprotein ligand-1 (PSGL-1), a glycoprotein expressed on the cell surface of leukocytes, binds to selectins and mediates leukocyte rolling on the vascular endothelium. Here we report that PSGL-1 binds to the C- terminal (G3 domain) of the extracellular proteoglycan PG-M/versican. Cells transfected with PSGL-1 or a shorter form containing the binding site, or cells expressing endogenous PSGL-1 aggregate in the presence of versican or G3 product. The aggregation appears to be induced by G3 multimers that bind to PSGL-1 and form a network. Endogenous versican and/or G3-containing fragments also bind to PSGL-1 in human plasma. Removal of the endogenous G3-containing fragments reduces the effect of plasma on leukocyte aggregation. Finally, the roles of G3-containing fragments in leukocyte aggregation were confirmed in a mouse model. Taken together, our results strongly support a physiologically relevant role for PSGL- 1/versican binding and may have implications in the immunoresponse. Key words: PSGL-1, Selectin, G3 domain, Interaction, Aggregation Summary PG-M/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation Peng-Sheng Zheng 1,3 , Dana Vais 1,3 , David LaPierre 1,3 , Yao-Yun Liang 2 , Vivian Lee 1,3 , Bing L. Yang 1,3 and Burton B. Yang 1,3, * 1 Sunnybrook and Women’s College Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada 2 Michael E. DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5G 1L5, Canada *Author for correspondence (e-mail: burton.yang@sw.ca) Accepted 31 August 2004 Journal of Cell Science 117, 5887-5895 Published by The Company of Biologists 2004 doi:10.1242/jcs.01516 Research Article