Amyloid Fibril Formation by Bovine Milk α s2 -Casein Occurs under Physiological Conditions Yet Is Prevented by Its Natural Counterpart, α s1 -Casein

Abstract

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 αs2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole αs-casein, αs2-casein was separated from αs1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37°C, the spherical particles typical of αs2-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, αs2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that αs2-casein fibril formation is potently inhibited by its natural counterpart, αs1-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.