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

Abstract

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.

Serum Albumin in Health and Disease: Esterase, Antioxidant, Transporting and Signaling Properties

“…As already mentioned, albumin is synthesized and enters the bloodstream as a non-glycosylated protein, but even in a healthy persons’ blood plasma, a certain proportion of albumin molecules undergo glycation over time, which can affect their structural and functional characteristics [ 155 , 156 ]. According to some reports, in normoglycemic blood 10 to 18% of circulating proteins are glycated in vivo, while in diabetic blood this proportion reaches up to 40% [ 131 , 157 ].…”

Antiglycation potential of plant based TiO2 nanoparticle in D‐ribose glycated BSA in vitro

“…The protein binding ANS is an extrinsic quantum fluorescent dye. The elevation in its fluorescence intensity depends upon hydrophobic nature of protein and ANS binding position particularly with a polar surface 43 . Hydrophobic‐residue‐specific‐ANS assay showed several fold increases in fluorescence intensity in case of glycated BSA comparative to native one.…”

“…The elevation in its fluorescence intensity depends upon hydrophobic nature of protein and ANS binding position particularly with a polar surface. 43 Hydrophobic-residue-specific-ANS assay showed several fold increases in fluorescence intensity in case of glycated BSA comparative to native one. Increase in quantum fluorescence intensity depicts conformational changes in its tertiary structure and structural transition from folded to unfolded state with increased exposition of hydrophobic residues to the solvent.…”

Understanding the Role of Protein Glycation in the Amyloid Aggregation Process

“…In particular, glycation has been shown to promote strong conformational changes in HSA that affect both secondary and tertiary structure and markedly reduce the protein stability. In this way, glycation promotes amyloid aggregation in HSA both reducing the helical content and supporting the formation of β-cross structure that rapidly evolve to the formation of amyloid aggregates [ 129 , 139 , 140 , 141 ]. Interestingly, glycation of albumin performed in the presence of D-ribose, besides promoting amyloid formation, has been shown to stabilize the amyloid oligomeric species that result highly cytotoxic in neuronal cells [ 129 ].…”