Amyloid Fibril Formation by β-Casein and Its Influence Factor†

doi:10.7503/cjcu20130873

Abstract

Abstract:

β-Casein is the second abundant among the casein proteins in bovine milk and reported to exhibite biological activities. In this study, the focus was placed on the influence of lipids and heparin sulphate to β-casein fibril formation. In order to study the time course of fibril formation by β-casein, the samples were incubated and picked up at specific times and tested by ThT assay and transmission electron microscopy. The results showed that amyloid fibrils were not formed by β-casein incubated in pH=5.4—9.0 at 65 ℃ for 252 h, which suggested that β-casein is a good molecular chaperone. The β-casein fibril formation was promoted in the presence of longer-chain phosphatidylcholine lipids(D6PC and D9PC), which indicated that the interaction of β-casein with biomembrane of mammary gland abundant with lipids maybe caused β-casein structure changed from native to more β-sheet. Heparin sulphate, a major component of the extracellular matrix and a species which is commonly associated with extracellular amyloid deposits, interacted with β-casein to promote its aggregation. It is supposed that Corpora Amylacea is associated with mastitis because of high expression of heparin sulphate in inflamed mammary. This study explored that it is possible for chaperon proteins to form amyloid fibrils influenced by components in vivo and lose its chaperon effects.

Key words: β-Casein, Amyloid fibril, Lipid, Heparin sulphate, ThT assay

CLC Number:

O629.73

TrendMD:

LIU Jihua, John A.Carver, David C. Thorn. Amyloid Fibril Formation by β-Casein and Its Influence Factor^†[J]. Chem. J. Chinese Universities, 2014, 35(5): 976.

Figures/Tables 6

Fig.1 ThT assay for β-casein incubated at 65 ℃ for 252 h in pH=5.4—9.0^a. pH=5.4; b. pH=5.8; c. pH=6.0; d. pH=6.5; e. pH=7.0; f. pH=8.0; g. pH=8.5; h. pH=9.0.

Fig.2 ThT assays for β-casein incubated with D6PC(A) and D9PC(B) at 65 ℃ for 252 h in pH=5.4^(A) a. 0.1 mmol/L; b. 0.3 mmol/L; c. 1 mmol/L; d. 3 mmol/L; e. β-CN;(B) a. 0.1 mmol/L; b. 1 mmol/L; c. 0.3 mmol/L; d. β-CN.

Fig.3 TEM images for β-casein incubated with 1.0(A) and 3.0 mmol/L(B) D6PC at 65 ℃ for 252 h in pH=5.4

Fig.4 TEM images for β-casein incubated with 0.1(A), 0.3(B) and 1.0 mmol/L(C) D9PC at 65 ℃ for 252 h in pH=5.4

Fig.5 ThT assays for effects of Heparin sulphate concentration to fibrils formation by β-casein^a. 5 μg/mL; b. 10 μg/mL; c. 20 μg/mL;d. 40 μg/mL; e. β-CN.

Fig.6 TEM images for effects of Heparin sulphate concentration to fibrils formation by β-casein^(A) 5 μg/mL; (B) 10 μg/mL; (C) 20 μg/mL; (D) 40 μg/mL.

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