Isolation of mitogenic glycophosphopeptides from cheese whey protein concentrate

We investigated the immunological function of cheese whey protein concentrate (CWPC), which is a by-product of cheese production, using mitogenic activity in murine splenocytes as an index. A fraction isolated by gel filtration and anion exchange chromatography of CWPC showed high mitogenic activity, comparable to the activity of lipopolysaccharide (LPS). The fraction was detected as a single band on SDS-PAGE. It contained calcium, inorganic phosphorus, and carbohydrate, indicating the active component to be a glycophosphopeptide (GPP). Since Pronase digestion of GPP did not reduce its mitogenic activity, carbohydrate rather than peptide may be important in the activity. When applied on an anti-beta-caseinophosphopeptide (beta-CPP) antibody affinity column, the GPP was separated into two components, one with affinity to beta-CPP and the other without such affinity. Both the components contained N-linked oligosaccharide chains and had the mitogenic activity. These results demonstrate that cheese whey contains a GPP having strong mitogenic activity.