Characterization of Heparin Interactions With Recombinant Rodent Stabilin-2/Hyaluronic Acid Receptor for Endocytosis (HARE).

Abstract

Stabilin-2 is the primary scavenger for hyaluronan (HA) and binds to over two dozen other ligands including chondroitin sulfates, heparin, oxidized/acetylated LDL, etc. Although rat liver sinusoidal endothelial cells are the preferred primary cell lines and animal for physiological studies of Stab2/HARE, the rat recombinant protein has never been characterized. Since the rat Stab2/HARE has a high degree homology to mouse Stab2/HARE which has been cloned, our hypothesis is that the rat receptor is identical to mouse and very similar to the human receptor. Rat Stab2/HARE was cloned and expressed in the FlpIn HEK293 cell line. The recombinant protein was analyzed for HA and heparin binding/endocytosis as well as synthetic heparin (Dekaparin) in a mouse knockout model. The secreted ecto-domain was also created for surface plasmon resonance analysis. The physical structure of rat Stab2/HARE is different than human in that the small isoform is not expressed as robustly and reduction of the protein results in what is likely two physical conformational forms. Rat Stab2/HARE binding strength with HA is weaker when compared to human Stab2/HARE, but rate of endocytosis is higher. Heparin-Stab2/HARE bonding strength is similar to human, though endocytic rate tends to be higher. Metabolism of Dekaprin is delayed in a Stab2KO mouse model and affects liver sequestration of this drug. Rat Stab2/HARE has similar properties as the human Stab2/HARE with the exceptions that the rat recombinant protein has a different physical structure and has an increased HA and heparin internalization rate.