Spectroscopic Study of Full-Length Recombinant Proteoglycan 4 (rhPRG4): Self-Assembly and Interactions with Hyaluronan

Abstract

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid (SF) and at the surface of articular cartilage. It functions as a critical cartilage boundary lubricant, both alone in a dose-dependent manner and synergistically with the repeating disaccharide hyaluronan (HA). The mechanism of the functional interaction with HA remains to be elucidated, but appears to be non-covalent in nature and be dependent on PRG4 structure and assembly. Furthermore, concentration of both PRG4 and HA, which can vary in disease, may affect the interaction as well. Recently, full-length recombinant human PRG4 (rhPRG4) has become available. The objective of this study was to characterize aggregation of rhPRG4 and investigate its interaction with HA using spectroscopic techniques.Spectroscopic techniques including uv absorbance, fluorescence, and light scattering, were employed to characterize the aggregation of rhPRG4 and interaction with HA over a range of physiological concentrations. Various dilutions of rhPRG4 in different buffers and their mixtures with different concentrations of HA, with and without other physiological molecules, were examined. rhPRG4 was provided by Lubris.UV absorbance and fluorescence spectroscopic data demonstrated concentration based aggregation and suggested a non-covalent linear aggregation of rhPRG4 molecules with increasing concentration. However, multiple species were also observed even at sub-physiological concentrations. Dynamic as well as multi-angle light scattering techniques suggested presence of species with various and large (∼MDa) molecular weights. These techniques also suggested the subtle interaction of PRG4 with HA can be influenced by sample preparation, storage conditions, and presence of other molecules.These results contribute to the understanding of rhPRG4's aggregation and interaction with HA, which can have functional consequences and therefore provides the framework for the development of potential new rhPRG4+HA containing biotherapeutics for the treatment of osteoarthritis and other conditions.