title: The Effect of Milk Constituents and Crowding Agents on Amyloid Fibril Formation by κ-Casein.
abstract: When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.
authors.affiliation: Pharmacy College, Jilin University , Changchun, Jilin Province 130021, China.|Department of Chemistry, School of Physical Sciences, The University of Adelaide , Adelaide, South Australia 5005, Australia.
authors.forename: Jihua
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Chemistry, School of Physical Sciences, The University of Adelaide , Adelaide, South Australia 5005, Australia.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Dehle
authors.affiliation: Department of Chemistry, School of Physical Sciences, The University of Adelaide , Adelaide, South Australia 5005, Australia.
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Research School of Chemistry, The Australian National University , Acton, Australian Capital Territory 2601, Australia.
authors.forename: Elmira
authors.initials: E
authors.lastname: Bahraminejad
authors.affiliation: School of Biological Sciences and Illawarra Health & Medical Research Institute, University of Wollongong , Wollongong, New South Wales 2522, Australia.
authors.forename: Heath
authors.initials: H
authors.lastname: Ecroyd
authors.affiliation: Research School of Chemistry, The Australian National University , Acton, Australian Capital Territory 2601, Australia.
authors.forename: David C
authors.initials: DC
authors.lastname: Thorn
authors.affiliation: Research School of Chemistry, The Australian National University , Acton, Australian Capital Territory 2601, Australia.
authors.forename: John A
authors.initials: JA
authors.lastname: Carver
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002942:Circular Dichroism; D007700:Kinetics; D008892:Milk / Q000737:chemistry*; D010084:Oxidation-Reduction; D013696:Temperature
chemical_list: D000682:Amyloid; D002364:Caseins
keywords: amyloid fibril formation; calcium chloride; heparan sulfate; lactose; macromolecular crowding; non-nucleation-dependent; phospholipid; thioflavin T; triglyceride; κ-casein