Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells

Abstract

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (−1)- and (−2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate

“…A crystallographic study of Hpse demonstrated the location of these anionic amino acid residues relatively inside of the enzyme molecule [ 53 ]. It is speculated that the negative charge itself can be repulsive for the binding of Hpse to HS, which is also supported by our previous physicochemical study [ 54 ]. Heparastatin (SF4), a compound optimized for inhibition of Hpse enzymatic activity, is a derivative of a bacterial product siastatin B that can inhibit the enzymatic activity of Hpse.…”

“…The binding of Hpse to immobilized heparin or GAG derived from MST cells was examined based on an ELISA-like method as described previously [ 49 , 54 ]. Briefly, streptavidin was immobilized onto the ELISA plate (Greiner, Frickenhausen, Germany, 655061).…”

“…The GAG was recovered as a precipitate of centrifugation (15,000 rpm, 30 min), solubilized with water, dialyzed, and lyophilized. The carboxyl group in the GAG structure was biotinylated using biotin-PEG4-hydrazide (B5578, Tokyo Chemical Industry, Tokyo, Japan) and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (E6383, Sigma-Aldrich) as stated elsewhere [ 49 , 54 ]. Disaccharide analysis [ 57 , 63 ] of the prepared GAG derived from MST cells indicated the composition as follows: ΔDi-0S, 31.1%; ΔDi-NS, 16.0%; ΔDi-6S, 7.4%; ΔDi-NS6S, 11.1%; ΔDi-2SNS, 2.8%; ΔDi-TriS, 31.6%.…”