Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk -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 highperformance 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 -casein (residues 106 to 169) [-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 -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 -casein-A(138-158) and its nonphosphorylated counterpartcasein-A(138-158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P) 149-casein-A(138-158) displayed growth-inhibitory activity against S. mutans (MIC, 59 g/ml [26 M]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.The caseins are the most abundant bovine milk proteins, and there are four major types: ␣ s1 -, ␣ s2 -, -, and -casein (23). All four caseins are phosphorylated on specific seryl residues, and in addition, -casein is glycosylated (4). -Casein is hydrolyzed by the enzyme chymosin between Phe 105 and Met 106 , generating two polypeptides: a hydrophobic N-terminal para--casein polypeptide -casein (residues 1 to 105) [-casein(1-105)] and a hydrophilic phosphorylated and glycosylated C-terminal polypeptide -casein(106-169), known as the caseinomacropeptide (CMP). CMP is heterogeneous and contains all the posttranslational modification sites (glycosylation and phosphorylation) of -casein. Six potential glycosylation sites have been identified on CMP (18), and up to five different carbohydrate moieties may be attached at each site (20). Three genetic variants of CMP have also been identified, originating from the precursors -casein A, B, and E, with variants A and B being the most common in bovine milk (15).CMP and CMP-derived peptides have been reported to have a variety of biological activities, such as suppression of gastric secretions (28), depression of platelet aggregation (2), inhibition of influenza virus hemagglutination (8), inhibition of cholera toxin binding (9), and immunomodulating activities (14). CMP has also been shown to incorporate into salivary pellicle and inhibit the adherence of Streptococcus mutans, the oral...
Kappacin, nonglycosylated -casein(106-169), is a novel antimicrobial peptide produced from -casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, -casein(138-158), of these forms, we have shown that the Asp 148 to Ala 148 substitution is responsible for the lesser antibacterial activity of -casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 M at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a -casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl 2 reduced bacterial viability by 3 log 10 and suppressed recovery of viability. In contrast 20 mM ZnCl 2 alone reduced bacterial viability by Ϸ1 log 10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.
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