Kappacin, a Novel Antibacterial Peptide from Bovine Milk

Abstract

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...

Structure-Activity Analysis of the Dermcidin-derived Peptide DCD-1L, an Anionic Antimicrobial Peptide Present in Human Sweat

“…Binding of magainin-1 to phospholipid vesicles, where the peptide self-assembles, inserts, and form largely pentameric pores, induces the leakage of cellular material. We speculate that in vivo, the channel formed by DCD-1L functions as a proton channel as suggested for kappacin, an AAMP from bovine milk (9,10). For kappacin it has been proposed that it forms ion channels with a proton influx at acidic pH, which slowly results in bacterial cell death by reducing intracellular pH (9).…”

“…Examples of AAMPs are bovine kappacin, the proenkephalinderived peptides peptide B and enkelytin, maximin H5 from the amphibian Bombina maxima, and lysenins from the earthworm Eisenia fetida (5,(7)(8)(9)(10). It has been suggested that these peptides may synergistically enhance the action of CAMPs or other antimicrobial factors in the first line of defense against microbial infection (11).…”

Geographic distribution of haplotype diversity at the bovine casein locus

“…The genes are organised in a cluster of approximately 250 kB [13,41] and share common transcription regulating elements [41]. The locus is considered to influence milk production traits [8,9,17,22,46] and antibacterial activities of derived peptides [29] may also affect the biological fitness of the offspring. Moreover, casein genes harbour a number of variants with suggested effects concerning traits such as the manufacturing properties of milk [1,35].…”

Caprine κ-Casein (CSN3) Polymorphism: New Developments in Molecular Knowledge

“…However, in contrast to the calcium-sensitive α s1 -, β-, and α s2 -caseins, the κ-casein fraction plays a crucial role in the formation, stabilization, and aggregation of the casein micelles and thus affects the technological (Mariani et al, 1976;Aleandri et al, 1990;Lodes et al, 1996;Falaki et al, 1997) and nutritional properties of milk (Mercier et al, 1973(Mercier et al, , 1976Malkoski et al, 2001). Therefore, the κ-casein gene (CSN3) is unlikely to be completely free of selective constraints (Ward et al, 1997).…”