Isolation and Characterization of a Cytotoxic Pentapeptide, κ-casecidin, from Bovine κ-casein Digested with Bovine Trypsin

Abstract

Bovine κ-casein produced cytotoxic activity toward mouse spleen cells when it was digested with bovine trypsin or chymotrypsin. The cytotoxic activity reached a maximum when the casein was digested with the trypsin at 45°C for 5h and was observed when the spleen cells were cultured for 6h with the digest. A cytotoxic peptide was isolated from a digest prepared from κ-casein treated with trypsin for 5h by a combination of ion-exchange column chromatography, gel filtration and reversed-phase high performance liquid chromatography. The sequence of the isolated peptide was estimated as Phe-Phe-Ser-Asp-Lys, being compatible with sequence 17-21 of bovine κ-casein. A chemically synthesized peptide, Phe-Phe-Ser-Asp-Lys, produced cytotoxic activity toward mouse spleen cells at a dose response manner. On the other hand, the isolated peptide retained cytotoxic activities toward animal cells such as mouse spleen T- and B-lymphocyte-enriched cells, bovine milk cells and Jurkat, Clone E6-1 cells (a cell line from human acute leukemic T-lymphocyte). The mouse lymphocytes cultured with the isolated peptide revealed an internucleosomal DNA fragmentation. The isolated peptide significantly inhibited the proliferative responses of mouse spleen cells stimulated by lipopolysaccharide, pokeweed mitogen, phytohemagglutinin and concanavalin A. Moreover, the isolated peptide showed notable antibacterial activity against Staphylococcus aureus IFO3060.