Amyloid Fibril Formation by Bovine Milk αs2-Casein Occurs under Physiological Conditions Yet Is Prevented by Its Natural Counterpart, αs1-Casein

• Published in: Biochemistry (Easton) 2008-03, Vol.47 (12), p.3926-3936
• Main Authors: Thorn, David C, Ecroyd, Heath, Sunde, Margaret, Poon, Stephen, Carver, John A
• Format: Article
• Language: English
• Subjects: Amyloid - biosynthesis; Animals; Caseins - isolation & purification; Caseins - metabolism; Cattle; Chromatography, Ion Exchange; Circular Dichroism; Female; Hydrogen-Ion Concentration; Microscopy, Electron, Transmission; Oxidation-Reduction; Temperature; Thiazoles - metabolism; Ultraviolet Rays; X-Ray Diffraction
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701278c

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.