Casein‐derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT‐29 tumor cells

Casein phosphopeptides β‐CN(1–25)4P and α s1 ‐CN(59–79)5P, from β‐ and α s1 ‐casein, respectively, both carrying the characteristic ‘acidic motif’ Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu, were chemically synthesized and administered to HT‐29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca 2+ ] i due to influx of extracellular Ca 2+ . The response was quantitatively higher with β‐CN(1–25)4P than α s1 ‐CN(59–79)5P. The synthetic peptide corresponding to the ‘acidic motif’ was ineffective and the dephosphorylated form of β‐CN(1–25)4P almost inactive. The lack of the N‐terminally located five amino acids, or sequence modifications within the N‐terminal segment of β‐CN(1–25)4P, caused a total loss of activity, whereas the lack of the C‐terminal segment preserved activity. In conclusion, the influx of calcium into HT‐29 cells caused by β‐CN(1–25)4P appears to depend on the phosphorylated ‘acidic motif’ and the preceding N‐terminal region.