title: Amyloid fibril formation by bovine milk alpha s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha 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 alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-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, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-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.
authors.affiliation: School of Chemistry and Physics, The University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: David C
authors.initials: DC
authors.lastname: Thorn
authors.forename: Heath
authors.initials: H
authors.lastname: Ecroyd
authors.forename: Margaret
authors.initials: M
authors.lastname: Sunde
authors.forename: Stephen
authors.initials: S
authors.lastname: Poon
authors.forename: John A
authors.initials: JA
authors.lastname: Carver
mesh_terms: D000682:Amyloid / Q000096:biosynthesis*; D000818:Animals; D052160:Benzothiazoles; D002364:Caseins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D005260:Female; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D010084:Oxidation-Reduction; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism; D014466:Ultraviolet Rays; D014961:X-Ray Diffraction
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D002364:Caseins; D013844:Thiazoles; C009462:thioflavin T