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Effect of O-glycans on the Structure and Viscosity of Intrinsically Disordered and Glycosylated Protein Lubricin
Synovial joints exhibit remarkable lubrication mechanisms, essential for the smooth articulation of bones. This intricate process is orchestrated by key biomolecular components, such as hyaluronic acid, lipids, aggrecan, and glycoproteins. Lubricin, characterized by its intrinsically disordered region and highly glycosylated structure, is part of these glycoproteins. Lubricin significantly contributes to reducing viscosity and ensuring the sustained efficacy of synovial joints. The protein comprises two globular end domains and an intrinsically disordered central domain. The end domains bind to cartilage surfaces, allowing the remaining protein to extend into the synovial fluid. The central domain, known as the mucin-like domain, is abundant in O-glycans and mainly responsible for lubricin functionality. The O-glycans constitute approximately 30-35\% of the entire lubricin structure. O-glycans play a pivotal role in enabling lubricin to fulfill its highly specialized function. Despite the recognized importance of O-glycans, a detailed understanding of their influence on the structure and viscosity of lubricin remains elusive. Using extensive molecular dynamics (MD) simulations, in this thesis we unveil the influence of O-glycans on the rheological and viscosity properties of lubricin. In the first part of this thesis, our focus was on understanding how O-glycans influence the structure of lubricin. To achieve this, glycosylated fragments of lubricin were generated and modeled, and a suitable force field for intrinsically disordered glycoproteins was prepared. Five different segments of lubricin, each with a length of 80 amino acids, were considered. Utilizing Monte Carlo sampling, six different glycosylated fragments were introduced for each segment. Equilibrium molecular dynamics simulations were then conducted to explore the role of glycans in the conformational properties of lubricin. The findings reveal that the presence of O-glycans induces a more extended conformation in fragments of the disordered region of lubricin, resulting in a stiffer structure, and enhancing the exposure of lubricin to solvent molecules. These changes in the lubricin structure are attributed to the electrostatic and steric interactions imposed by the bulky side chains of O-glycans. The second phase of our study focused on unraveling the influence of glycans on the viscoelastic behavior of liquid systems containing lubricin fragments. Specifically, we aimed to understand how the solution viscosity may have been affected by the presence of this O-glycosylated protein. To achieve this, we employed the Green-Kubo method, i.e. we derived the zero-shear viscosity from the fluctuations of the pressure tensor in equilibrium molecular dynamics simulations. In addition, we carried shear-driven non-equilibrium molecular dynamics simulations to determine the viscosity under shear. Our simulations reveal that, unlike pure water, systems containing lubricin display a pronounced shear-thinning response. Furthermore, our findings demonstrate that glycosylation and the mass density of lubricin chains play a crucial role in regulating the system viscosity and its response to shear. Increasing mass density leads to higher viscosity, but the presence of O-glycans results in a reduction in solution viscosity and weakens shear thinning at high shear rates, compared to non-glycosylated systems with the same density. The electrostatic and steric interactions of O-glycans prevent the conglomeration and structuring of lubricin fragments, thereby altering the viscoelastic properties of lubricin. The results from our computational study provide a mechanistic understanding of previous experimental observations of lubricin, offering a more rational comprehension of its function in the synovial fluid.
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500 Naturwissenschaften und Mathematik
500 Natural sciences and mathematics