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

Casein phosphopeptides beta-CN(1-25)4P and alpha(sl)-CN(59-79)5P, from beta- and alpha(si)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-GluGlu, 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 [Ca2+](i) due to influx of extracellular Ca2+. The response was quantitatively higher with beta-CN(1-25)4P than alpha(si)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif was ineffective and the dephosphorylated form of beta-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 beta-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 beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.