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Kappacin, a novel antibacterial peptide from bovine milk
M Malkoski1, S G Dashper, N M O'Brien-Simpson
1School of Dental Science, The University of Melbourne, Melbourne, Victoria 3000, Australia.
Abstract:
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.
Insights
Caseinomacropeptide (CMP) from bovine milk exhibits antibacterial properties. The phosphorylated peptide kappacin (kappa-casein(106-169)) is identified as the active form, crucial for inhibiting oral pathogens like Streptococcus mutans.
Area of Science:
- Biochemistry
- Microbiology
- Food Science
Background:
- Caseinomacropeptide (CMP), a fragment of bovine kappa-casein, is known to be heterogeneous, existing in glycosylated and phosphorylated forms.
- Oral opportunistic pathogens such as Streptococcus mutans and Porphyromonas gingivalis, as well as Escherichia coli, pose significant health challenges.
- Previous research suggests potential bioactivities of CMP, necessitating further investigation into its specific components and mechanisms.
Purpose of the Study:
- To identify the specific active component within CMP responsible for growth-inhibitory activity against oral pathogens.
- To characterize the structural and chemical properties essential for this antibacterial activity.
- To investigate the role of phosphorylation in the bioactivity of CMP-derived peptides.
Main Methods:
- Fractionation of CMP using reversed-phase high-performance liquid chromatography (RP-HPLC).
- Antibacterial assays against Streptococcus mutans using isolated CMP fractions.
- N-terminal sequencing and mass spectrometry for fraction characterization.
- Enzymatic hydrolysis of CMP using Endoproteinase Glu-C, followed by peptide separation and activity testing.
- Chemical synthesis and antibacterial evaluation of specific phosphorylated and non-phosphorylated peptides.
Main Results:
- The nonglycosylated, phosphorylated kappa-casein (residues 106 to 169), designated kappacin, was identified as the active antibacterial moiety.
- The peptide Ser(P)(149)kappa-casein-A(138--158) was the sole active peptide generated by Endoproteinase Glu-C digestion.
- Synthetic Ser(P)(149)kappa-casein-A(138--158) demonstrated significant growth inhibition against S. mutans (MIC, 59 microg/ml), while its non-phosphorylated counterpart showed no activity.
Conclusions:
- Phosphorylation of kappa-casein(106-169) is essential for its antibacterial activity against Streptococcus mutans.
- Kappacin represents a promising antimicrobial peptide derived from bovine milk with potential applications in oral health.
- The findings highlight the therapeutic potential of milk-derived peptides in combating bacterial infections.