Bovine Serum Albumin Amyloid Fibrosis And Its Cellular Toxicity

Proteins and polypeptides are able to form amyloid fibrils which have been characterized to be rich in (3-sheet structures and toxic to biological systems. Substantial reports showed that amyloid fibillation is a common property of proteins. To date, more than 30 proteins are proved to have the ability to form fibrils which are related with human diseases, such asβ-amyloid peptides, prion, lysozyme, insulin and a-synuclein. The amyloidogenesis of proteins and peptides resulted in organ damages and dysfunctions and eventually cause diseases, including typeⅡdiabetes, Alzheimer's disease, Parkinson's disease, hemodialysis-related amyloid deposition and a number of systemic amyloidoses. Usually amyloid fibrils form and deposit in the vicinity of the target cells and accumulate on the cell membrane, triggering a cascade of cellular events which lead to apoptosis.Amyloid formation is a complex process and proceeds through a series of heterogeneous intermediates of various sizes and morphologies, including oligomers, protofilaments and mature fibrils. Oligomers and protofilaments have been identified as the main species that are responsible for the cytotoxicity of amyloid fibrils, although in some cases, denatured monomers, non-fibrillar or amorphous aggregates and mature fibrils also exhibit cytotoxicity. However, the accurate mechanism of amyloidogenesis is still not clear. It is essential to elucidate the cytotoxic mechanism of amyloid fibrils for developing novel medicines for the treatment of amyloid diseases.In this study, a non-pathogenic protein, BSA (bovine serum albumin) has been chosen as a model molecule to explore the conditions of fibrillation and the fibrillar toxicity of a protein with high molecular weight. Amyloid fibrils of lysozyme and insulin have been utilized as positive control to compare the growing kinetics, fibrillar morphologies, physical properties and cytotoxicities. The goal of this study is to elucidate whether the size or molecular weight is a key factor to which a protein assembly into a fibrillar structure with cytotoxicity.Experimental methods and results:1,Growing kinetics of BSA fibrillationThT is a fluorescent probe which can bind specifically toβ-sheet structure of a protein and therefore has been used to monitor the growth of amyloid fibrils. The results showed that the content ofβ-sheets was increased along the duration of incubation of BSA, indicating a formation of fibrillar structure. The growth curve of BSA fibrils did not appear as a sigmoidal shape as that of insulin fibrils and lysozyme fibrils.2,Surface hydrophobicity of BSA fibrils probed by ANS fluorescence ANS is a kind of fluorescence dyes that combines with hydrophobic domains of a protein and the fluorescence intensity is related to the surface hydrophobicity of BSA assembly. Results showed that in the process of BSA fibrillation, ANS intensity is decreasing with incubation time and without blue shift, an phenomena opposite to the tendencies of amyloid fibrillation of insulin and lysozyme.3,Circular dichroism results of BSA fibrillationCircular dichroism (CD) is a tool to detect secondary structure changes of proteins. It has been observed that a-helix decreased andβ-sheet increased upon the formation of BSA fibrils, similar to that of insulin fibrils and lysozyme fibrils.1,AFM and TEM images of BSA fibrilsAFM and TEM are common technologies to acquire the morphology of fibrils. The mature BSA fibrils were observed under AFM and TEM. The results demonstrated the BSA fibrils had a net-like fibrillar structure. In contrast, the insulin fibrils and lysozyme fibrils showed a long and non-branched morphology.2,SDS-PAGE to determine the interaction of BSA fibrils with cellular membranesMembranes of human red blood cells were incubated with amyloid fibrils prior to SDS-PAGE. The results showed that the spectrum of protein bands was not changed by BSA fibrils; while aggregation of cytoskeleton proteins occurred in the membrane samples pretreated by insulin fibrils and lysozyme fibrils. The results implicated that BSA fibrils lacked the ability to disrupt cellular membranes.3,Hemolytic assaySimilar as the results of SDS-PAGE, BSA fibrils did not affect the intact of red blood cells. In contrast, insulin fibrils and lysozyme fibrils induced strongly hemolysis of the cells.In conclusion, BSA is able to assembly into amyloid fibrils upon incubation. The resultant fibrils had typical fibrillar properties, including increased content ofβ-sheet structures, ThT binding and a net-like fibrillar structure. In contract with the amyloids of insulin and lysozyme, BSA fibrils showed lower surface hydrophobicity than its native monomers and therefore lacked the ability to disrupt cellular membranes. We concluded that a protein with high molecular weight such as BSA can also form amyloid fibrils, although the resultant amyloids showed no cytotoxicity due mainly to their low surface hydrophobicity.