The role of glycosylation in amyloid fibril formation of bovine κ-casein

In order to explore the functions of glycosylation of kappa-Casein (kappa-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of kappa-CN B were purified by selective precipitation followed by anion exchange chromatography from kappa-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled kappa-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0-28.7 nm range for a 1 mg mL(-1) solution. The presence of two bound glycans in the protein structure of 2G kappa-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 +/- 29.9 vs 50.3 +/- 13.7 nm) and width (8.6 +/- 2.1 vs 13.9 +/- 2.5 nm) for fibril morphology compared to UG kappa-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric kappa-CN B proteins at concentrations of 0.22-0.44 mg mL(-1). However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated kappa-CN present in a micelle-like form or dissociated monomeric kappa-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of kappa-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG kappa-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of kappa-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.