Kappacin, a Novel Antibacterial Peptide from Bovine Milk

M Malkoski

S Dashper

N O'Brien-Simpson

G Talbo

M Macris

K Cross

E Reynolds

School of Dental Science, The University of Melbourne, Melbourne, Victoria 3000,^{and Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Melbourne, Victoria 3101,}^Australia

^{Corresponding author. Mailing address: School of Dental Science, The University of Melbourne, 711 Elizabeth St., Melbourne, Victoria 3000, Australia. Phone: 61 3 9341 0270. Fax: 61 3 9341 0236. E-mail:}ua.ude.bleminu.tned@sdlonyer.e.

Received 2000 Sep 18; Revisions requested 2001 Jan 30; Accepted 2001 May 9.

Abstract

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-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 κ-casein (residues 106 to 169) [κ-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)^{κ-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)^{κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(}P)^{κ-casein-A(138–158) displayed growth-inhibitory activity against}S. mutans (MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Abstract

The caseins are the most abundant bovine milk proteins, and there are four major types: α_s1-, α_s2-, β-, and κ-casein (23). All four caseins are phosphorylated on specific seryl residues, and in addition, κ-casein is glycosylated (4). κ-Casein is hydrolyzed by the enzyme chymosin between Phe^{and Met}^{, generating two polypeptides: a hydrophobic N-terminal para-κ-casein polypeptide κ-casein (residues 1 to 105) [κ-casein(1–105)] and a hydrophilic phosphorylated and glycosylated C}-terminal polypeptide κ-casein(106–169), known as the caseinomacropeptide (CMP). CMP is heterogeneous and contains all the posttranslational modification sites (glycosylation and phosphorylation) of κ-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 κ-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 pathogen implicated in the development of dental caries (12, 17, 21, 25, 26). The incorporation of CMP into salivary pellicle is proposed to be the mechanism by which certain milk protein fractions, when added to the cariogenic diet of rats, substantially reduce caries activity and the recovery of mutans group streptococci from the experimental animals (7). Examination of the amino acid sequence of CMP revealed that the peptide contains residues that could form an amphipathic helical structure, and as such, the peptide may possess antibacterial properties.

Antibacterial peptides have been isolated and characterized from mucosal surfaces of the gastrointestinal tract and secretions of a wide variety of organisms (2, 13). In general, these peptides have been reported to contain a high percentage of basic amino acyl residues in an amphipathic structure. These characteristics have been proposed to facilitate interaction between the positively charged peptide and the negatively charged bacterial membrane (13). Antibacterial peptides have been characterized according to their size, conformation, and amino acid composition into α-helical amphipathic peptides, cysteine-rich β-sheet amphipathic peptides, disulfide ring peptides, linear peptides with proline-rich sequence motifs (13), and, more recently, phosphorylated and glycosylated anionic antibacterial peptides (22).

We present here evidence that CMP has activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. Fractionation of CMP, using reversed-phase high-performance liquid chromatography (RP-HPLC), revealed that the nonglycosylated, phosphorylated form of κ-casein(106–169), which we have designated kappacin, was the active form of the peptide against S. mutans. Hydrolysis of CMP with endoproteinase Glu-C generated a range of peptides, of which only Ser(P)^{κ-casein(138–158) exhibited growth-inhibitory activity. Peptides corresponding to Ser(}P)^{κ-casein-A(138–158) and its nonphosphorylated counterpart, κ-casein-A(138–158), were chemically synthesized and tested for antibacterial activity. The phosphorylated peptide Ser(}P)^{κ-casein-A(138–158) exhibited growth-inhibitory activity against}S. mutans (MIC, 59 μg/ml [26 μM]); however, the nonphosphorylated peptide did not inhibit growth of the bacterium, indicating that phosphorylation is essential for antibacterial activity.

ACKNOWLEDGMENTS

M.M. is the recipient of an Australian Dairy Research Development Corporation postgraduate research scholarship. The financial support of the Australian Industry R&D Board, the Victorian Dairy Industry Authority, and Bonlac Foods Ltd is gratefully acknowledged.

We thank Peter Riley for N-terminal sequence analysis and Rita Paolini, Paul Veith, David Eakins, and Daniela Salvatore for their excellent technical assistance.

ACKNOWLEDGMENTS

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