Purification, partial characterization of rat kidney hyaluronic acid binding protein and its localization on the cell surface

Abstract

Hyaluronic acid binding protein (HABP) has been purified to homogeneity from normal adult rat kidney by hyaluronate Sepharose affinity chromatography, and its apparent molecular mass was found to be 68 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of HABP under reducing as well as nonreducing conditions revealed a single protein band of 34 kDa, thus indicating that kidney HABP is a homodimer and lacks interchain disulfide bond. Its glycoprotein nature was demonstrated by Con-A binding analysis. The pI value of kidney HABP was 6, indicating its acidic nature. Polyclonal antibodies were raised against it, and the monospecificity of the antibodies towards HABP was confirmed by Western blot analysis of tissue extracts. Immunoblot analysis has elucidated the occurrence of this glycoprotein in various tissues. Moreover, HABP present in these tissues are shown to be structurally and immunologically identical. However, this glycoprotein is antigenically distinct from other well characterized extracellular proteins, e.g., fibronectin, laminin and collagen type IV. With the help of enzyme-linked immunosorbent assay (ELISA) and iodinated [125I]HABP, it has been shown that kidney HABP binds specifically to hyaluronic acid (HA) amongst all the glycosaminoglycans (GAGs), however, HABP can interact with other matrix proteins, e.g., laminin, fibronectin, and collagen type IV. The apparent dissociation constants of HABP for HA, laminin, fibronectin, and collagen type IV were approximately in the range of 10(-9) M, and kinetic analysis showed that these binding interactions were complex and of positive cooperative nature. Indirect immunofluorescence staining demonstrated its localization on human fetus lung fibroblast cell surface. Detection of 34 kDa HABP in the serum-free supernatant culture medium of fibroblasts was further evident by immunoblot analysis, thus confirming the secretory nature of HABP and its occurrence in the extracellular matrix.