Malkoski M, Dashper S G, O'Brien-Simpson N M, Talbo G H, Macris M, Cross K J, Reynolds E C
School of Dental Science, The University of Melbourne, Melbourne, Victoria 3000, Australia.
Antimicrob Agents Chemother. 2001 Aug;45(8):2309-15. doi: 10.1128/AAC.45.8.2309-2315.2001.
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.
酪蛋白巨肽（CMP）是牛乳κ-酪蛋白的一种异质性C末端片段（第106至169位氨基酸残基），由不同基因变体的糖基化和磷酸化形式组成。我们已经证明，CMP对口腔机会致病菌变形链球菌、牙龈卟啉单胞菌以及大肠杆菌具有生长抑制活性。使用反相高效液相色谱法（RP-HPLC）对CMP进行分级分离，并在96孔板肉汤试验中检测各馏分对变形链球菌的活性。通过N末端序列分析和质谱对馏分进行表征。结果表明，CMP的活性形式为非糖基化、磷酸化的κ-酪蛋白（第106至169位氨基酸残基）[κ-酪蛋白（106-169）]，我们将其命名为κ-酪蛋白素。使用内肽酶Glu-C水解CMP，生成的肽段通过RP-HPLC和凝胶过滤-HPLC进行分离，然后检测其对变形链球菌的活性。肽段Ser(P)(149)κ-酪蛋白-A(138-158)是内肽酶Glu-C消化产生的唯一具有生长抑制活性的肽段。化学合成了与抑制性肽段Ser(P)(149)κ-酪蛋白-A(138-158)及其非磷酸化对应物κ-酪蛋白-A(138-158)序列相应的肽段，并检测其抗菌活性。合成的Ser(P)(149)κ-酪蛋白-A(138-158)对变形链球菌具有生长抑制活性（最低抑菌浓度，59μg/ml [26μM]）。然而，非磷酸化肽段在所测试的浓度下没有抑制生长，这表明磷酸化对于活性至关重要。
