Hyaluronan receptors of liver endothelial cells, their purification and characterisation

Sammanfattning: Hyaluronan (HA) is a polysaccharide common throughout the connective tissues. and is found at low concentrations in the blood. The major site of its clearance is via an endocytic receptor on liver endothelial cells (LEC). This process is vital, as excessive serum levels of HA lead to clotting disorders and overly viscous blood. This thesis details studies towards the identification and characterisation of this receptor. A 100 kDa protein, previously identified as a candidate HA receptor on LEC using HA-Sepharose chromatography, was purified, sequenced and identified as ICAM-1. However, examination of its affinity for HA-Sepharose suggested that it actually interacted with blocked diaminohexane based linkers that would otherwise couple HA to Sepharose, and not with the HA itself. Furthermore, it was found that HA also had an affinity for the same blockedlinkers, and could displace ICAM-1 from them. The HA-binding moiety was identified as l-amino-6-acetoamidohexane, which may serve as a useful aid in the study of HA and its interactions with other bio-molecules.In the search for a means of purifying the LEC HA receptor, it was found that HA coupled to Sepharose via a shorter ethylenediamine based linker could specifically purify two HA binding proteins of approximately 200 and 400 kDa from the surface of LEC. These proteins were also found to have an affinity for scavenger receptor (SR) ligands, and have been purified on a large scale. Polyclonal antibodies to these proteins do not stain culturedhepatocytes, but do stain endocytic vesicles in cultured LEC, and specifically inhibit their uptake of HA and some scavenger receptor ligands. The potential role of the HA receptor in the clearance of SR ligands may have implications for diseases involving the pathological deposition of SR ligands.