Studies of Glycosaminoglycan Interactions - Surface Immobilization Strategies and Biosensing Applications

Doctoral thesis, 2014

Many important biological functions of glycosaminoglycans (GAGs) have been highlighted in research literature during recent years. GAGs often serve as function-bearing structural elements in the extracellular matrix (ECM), but are also constituents of the cell membrane. GAGs take part in various biological mechanisms, e.g. regulating tissue growth and maintenance, as well as in the development of different diseases. The varying chemical structure of GAGs promotes interesting properties, but also makes them challenging to study. Surface-based analytical techniques can provide detailed information about the many interactions that GAGs participate in. This requires immobilization of one of the interacting entities in a biofunctional manner to ensure reliability of the subsequent interaction studies. In this thesis, different methods for immobilizing GAGs to surfaces were investigated with the aim of studying GAG-related interactions. The GAGs chondroitin sulfate (CS) and hyaluronan (HA), as well as synthetically sulfated derivatives were primarily used. Immobilization was made to supported lipid bilayers on silica and self-assembled monolayers on gold, either using native GAGs or variants functionalized with e.g. biotin. The formation of GAG-based layer-by-layer assemblies was also studied. Immobilization and subsequent interactions were followed in real-time using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR)-based sensing. Interaction studies with various biological entities were made. The interactions were highly dependent on the orientation of the GAGs on the surface, and pros and cons associated with side-on versus end-on immobilization are discussed in the thesis. The immobilization strategy, especially if functional groups were introduced on the GAG, also influenced how the GAG was recognized by an interacting protein. Further, sulfated GAGs are known to serve as attachment factors for certain viruses, and binding studies of a herpes simplex virus glycoprotein to immobilized GAGs revealed interesting characteristics of certain regions on the protein. Also the initial effect of chondrocytes on immobilized HA was studied using combined surface sensing and light microscopy. The results presented here emphasize important aspects to consider when designing GAG-based interaction platforms, and exemplifies important biological studies that can be made by utilizing such platforms.