Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, αS2- and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (αS1 and β) as well as β-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow αS1- and β-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, αS1- and β-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel β-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel β-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of β-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly; in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins diversified partly to avoid dysfunctional amyloid fibril formation.
Keywords: Amyloid fibril; Amyloidogenic sequence; Functional amyloid; Intrinsically disordered protein; Oligomer; Polyproline-II helix; Proline/glutamine-rich; alphaS1-casein; beta-casein.
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