The impact of three amino acids with distinct electrical properties on the synthesis of α-casein by MAC-T cells

Abstract

This study utilized in vitro cultured bovine mammary epithelial cells line(MAC-T cells) to examine how different concentrations of non-polar proline (Pro), polar negatively charged glutamic acid (Glu), and polar uncharged serine (Ser) affect α-casein synthesis. A multi-factor level completely randomized experimental design was adopted. MAC-T cells were cultured in vitro using a medium containing 10% fetal bovine serum (10% FBS DMEM). After adhering to the wall, the cells were starved in serum-free medium for 12 h. The cells in the serum-free medium group were used as the blank control group (CK, 0× group), and the experimental groups were respectively supplemented with Pro (0.52, 1.04, 2.09, 4.17, 8.35, 16.69 mmol/L), Glu (0.77, 1.53, 3.06, 6.13, 12.25, 24.50 mmol/L), and Ser (0.50, 0.99, 1.97, 3.93, 7.86, 15.71 mmol/L). The proliferation rate of MAC-T cells was detected by CCK8 method; the synthesis amount of α-casein inside and outside MAC-T cells was detected by enzyme-linked immunosorbent assay; the expression levels of genes related to the synthesis of α-casein in MAC-T cells were detected by real-time fluorescence quantitative PCR (RT-qPCR) technology. The addition of Glu had the highest proliferation rate and the most significant effect at 12 h. The addition of 16.69 mmol/L Pro, 24.50 mmol/L Glu, and 0.99 mmol/L Ser could significantly increase the synthesis of α-casein (P < 0.01). When Pro, Glu, and Ser were added respectively, they all promoted the up-regulation of the expression of α-casein synthesis-related genes CSN1S1 and CSN1S2 in MAC-Tcells. Amino acids with distinct charge natures (acidic Glu, polar Ser, non-polar Pro) all exhibited positive regulatory roles in the synthesis of α-casein in MAC-T cells. Among them, the non-polar amino acid proline had the most pronounced promoting effect, disclosing that the disparity in charge characteristics is a crucial regulatory factor affecting the synthesis of α-casein.