Amyloid fibril formation by bovine milk α_s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, α_s1-casein

The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk α_s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole α_s-casein, α_s2-casein was separated from α_s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37°C, the spherical particles typical of α_s2-casein rapidly converted to twisted, ribbon-like fibrils ∼12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a β-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, α_s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that α_s2-casein fibril formation is potently inhibited by its natural counterpart, α_s1-casein, while is only partially inhibited by β-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.