Ribosylation induced structural changes in Bovine Serum Albumin: understanding high dietary sugar induced protein aggregation and amyloid formation.

Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.