Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Abstract

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.

Identification of the Phosphorylated Sites of Metabolically 32P-Labeled Osteopontin from Cultured Chicken Osteoblasts

“…The unique cleavage of OPN by thrombin (19,(53)(54)(55)(56)(57), which cleaved chicken OPN at two specific sites (one between Arg-22, and Ser-23 and the other between Lys-138 and Ala-139) (Fig. 6), was used to establish that phosphorylated sites were distributed throughout the length of the OPN molecule, which is synthesized and secreted by cultured chicken osteoblasts.…”

“…These results were based on calculations from the HPLC-purified material in terms of the 32 P counts and protein content. Furthermore, from such data the calculated ratio of 32 P label/OPN protein for medium and mineralized layer were comparable, suggesting that the phosphorylated forms of OPN in these two compartments were of very similar nature.The unique cleavage of OPN by thrombin (19,(53)(54)(55)(56)(57), which cleaved chicken OPN at two specific sites (one between Arg-22, and Ser-23 and the other between Lys-138 and Ala-139) (Fig. 6), was used to establish that phosphorylated sites were distributed throughout the length of the OPN molecule, which is synthesized and secreted by cultured chicken osteoblasts.…”

The Role of Osteopontin in Tumor Progression and Metastasis in Breast Cancer

“…Fragments of osteopontin originating from either unknown or other proteolytic activities have also been identified in human milk (67) and in human uterus (91). Functionally, fragments of osteopontin produced by thrombin cleavage amplify the effects of the full-length protein (92). For example, a variety of human cell lines exhibit more extensive cell attachment and spreading on thrombin-cleaved osteopontin compared with uncleaved osteopontin (92).…”

“…Functionally, fragments of osteopontin produced by thrombin cleavage amplify the effects of the full-length protein (92). For example, a variety of human cell lines exhibit more extensive cell attachment and spreading on thrombin-cleaved osteopontin compared with uncleaved osteopontin (92). The receptor on the cells mediating the attachment is the a v h 3 integrin, introducing this receptor as a major functional receptor for thrombin-cleaved osteopontin.…”

“…The receptor on the cells mediating the attachment is the a v h 3 integrin, introducing this receptor as a major functional receptor for thrombin-cleaved osteopontin. The cleavage of osteopontin may change the conformation of the molecule, more specifically, the conformation of the sequence around the RGD motif, and thereby affect the binding to the a v h 3 integrin by allowing greater accessibility to the receptor (92). The RGD motif is contained in the NH 2 -terminal fragment of thrombincleaved osteopontin and it is this fragment that promotes a stronger response (ref.…”