Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus-transformed 3T3 cells

Abstract

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyaluronan-mediated motility: a target in oral squamous cell carcinoma

“…Preliminary in vitro studies have suggested potential roles for two molecularly distinct cell-surface receptors for HA, i.e., RHAMM and CD44 play pivotal roles in tumor invasion and metastasis (21,22). RHAMM was isolated originally from culture supernatants of ras-transformed murine 3T3 fibroblasts (23). In addition to its extracellular localization, RHAMM also has been shown to be an intracellular protein associated with extracellular-regulated kinase (ERK) and it plays an important role in transforming the activity of H-ras in fibroblasts (24).…”

Requirement of the hyaluronan receptor RHAMM in neurite extension and motility as demonstrated in primary neurons and neuronal cell lines

“…Since HA binding domains are found in other ECM proteins including neurocan, versican/ CHAP, hyaluronectin, and aggrecan (Hardingham and Fosang, 1992;Laurent and Fraser, 1992;Rauch et al, 1992) it is conceivable that interference of HA binding to RHAMM alters HA interactions with other HA binding proteins. Conversely, RHAMM has been shown to bind to heparin, laminin, and collagen type IV, albeit with lower affinity than it binds to HA (Turley et al, 1987). Thus, anti-RHAMM antibodies and RHAMM peptides may affect neurite migration, in part, by interfering with interactions of RHAMM with other ECM components shown to affect neurite outgrowth (Carbonetto et al, 1983;Bozyczko and Horwitz, 1986;Carri et al, 1988;Dow et al, 1993).…”

Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid

“…Hyaluronic acid (HA) is an abundant component of the extracellular matrix. Three receptors for HA on the cell surface, namely CD44 (Aru o et al, 1990), RHAMM (Turley et al, 1987) and LYVE (Banerji et al, 1999), have been identi®ed. CD44, the principal cellular surface receptor for hyaluronic acid, comprises a family of many isoforms (85 ± 200 kDa) that arise through alternative splicing of the preRNA and di erent degrees of glycosylation (Naor et al, 1997).…”