CD44 and Annexin A2 Mediate the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein

Abstract

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5adesArg (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.

Upregulation of Vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules

“…Plasma-derived DBP also binds to the surface of many cell types including neutrophils (DiMartino and Kew, 1999;DiMartino et al, 2001;White and Cooke, 2000). DBP appears to bind with low affinity to multiple cell surface ligands such as chondroitin sulfate proteoglycans (DiMartino and Kew, 1999), megalin (Nykjaer et al, 1999;Nykjaer et al, 2001), cubulin (Nykjaer et al, 2001), CD44 and annexin A2 (McVoy and Kew, 2005). Neutrophils transiently generate co-chemotactic activity for C5a on the cell surface within 15-20 min of DBP binding (Kew et al, 1995a).…”

Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3

“…AP on the external face of the plasma membrane also has been shown to associate with annexin A2 in lipid rafts (Gillette and Nielsen-Preiss, 2004). Moreover, our lab has recently shown that annexin A2 may serve as part of the DBP cell surface binding site (McVoy and Kew, 2005). These studies have provided the rationale to investigate if there is an association between 1,25(OH) 2 D 3 and AP.…”

“…Several studies from our laboratory have demonstrated that the binding of DBP to the neutrophil plasma membrane, and subsequent protease-mediated shedding of the binding site, are essential for the chemotaxis enhancement of C5a (DiMartino and Kew, 1999;DiMartino et al, 2001;Kew et al, 1995a;Kew et al, 1995b) More recently, we have demonstrated that DBP requires platelet-derived thrombospondin-1 for maximal cochemotactic activity (Trujillo and Kew, 2004). In addition, cell surface CD44 and annexin A2 are part of a DBP binding site complex and mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005). The current study provides evidence that the putative cellular binding site on DBP is not altered by ligation with vitamin D sterols or G-actin.…”

“…Both plasma membrane binding and subsequent shedding of DBP are essential for the protein to function as a chemotactic cofactor for C5a. Recent studies from our lab also have shown that DBP requires platelet-derived thrombospondin-1 for maximal co-chemotactic activity (Trujillo and Kew, 2004), and that cell surface CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005 …”

“…Annexin A2 is a widely expressed member of a family of Ca 2+ -dependent phospholipid binding proteins that has been shown to facilitate formation of signaling complexes on both the external and cytoplasmic faces of the plasma membrane (Merrifield et al, 2001). Our lab has recently shown that annexin A2 serves as part of the DBP cell surface binding site (McVoy and Kew, 2005). Interestingly, AP on the external face of the plasma membrane also has been shown to associate with annexin A2 in lipid rafts (Gillette and NielsenPreiss, 2004).…”