Kappacin, a novel antibacterial peptide from bovine milk.
Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.
Malkoski
M
M
School of Dental Science, The University of Melbourne, Melbourne, Victoria 3000, Australia.
Dashper
S G
SG
O'Brien-Simpson
N M
NM
Talbo
G H
GH
Macris
M
M
Cross
K J
KJ
Reynolds
E C
EC
D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D002364:Caseins / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology*; D010766:Phosphorylation; D016966:Porphyromonas gingivalis / Q000187:drug effects* / Q000254:growth & development; D013295:Streptococcus mutans / Q000187:drug effects* / Q000235:genetics
D000900:Anti-Bacterial Agents; D002364:Caseins; D010446:Peptide Fragments; C065989:caseinomacropeptide