Bacteriostatic Activity of Whey Acidic Protein (WAP)

Abstract

ABSTRACT. We have previously reported the action of whey acidic protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls. The whey acidic protein (WAP) with a four-disulfide core (4-DSC) structure (WAP motif) composed of eight cysteine residues has been identified in milk in a range of species [1,4,6,7,9,11,15]. Possible functions of WAP have been proposed based on its structural similarity to protease inhibitor [9]. However, only a few studies have attempted to determine the actual function of WAP [3,20]. We have previously reported that WAP inhibits the proliferation of mouse mammary epithelial cells in vivo and in vitro [12,[14][15][16][17] and has a function in reducing tumorigenesis and invasion of mammary cancer cells [18]. A number of antibacterial proteins with a WAP motif have been identified in a variety of tissues,-e.g., elafin [22], eppin [24], SLPI [25], SWAM1 and SWAM2 [10], although the molecular antibacterial mechanism of these proteins is not entirely clear. On the basis of homologous amino acid sequences in the WAP motif between WAP and known antibacterial proteins with WAP motifs, WAP may also have antibacterial potential. However, because the amino acid sequences of WAP motif proteins are widely divergent, with the exception of the conserved cysteine residues, it is quite difficult to predict their antibacterial activity merely by examining these sequences.In the present study, we investigated the antibacterial activity of WAP because there have been no studies investigating the effect of WAP on microbes.Milk was collected from lactating Wistar-Imamichi rats (Japan SLC, Hamamatsu, Japan) as described by Simoms et al. [21]. The viscous milk was diluted 1:3 with chilled distilled water, and fat was removed by centrifugation for 30 min at 3,100  g at 4C. The supernatant was adjusted to pH 4.6, and caseins were removed by centrifugation for 30 min at 3,100  g at 4C. The supernatant was adjusted to pH 7.0, and was then loaded on a Sephadex G-75 column (2.6  90 cm; Invitrogen, Carlsbad, CA, U.S.A.) equilibrated in 50 mM Tris-HCl (pH 7.0)/10 mM NaCl. Four milliliters of the eluate was collected at a flow rate of 0.2 ml/min at 4C and dialyzed overnight at 4C against distilled water. A part of each eluate was examined spectrophotometrically (SmartSpecTM Plus; Bio-Rad, Hercules, CA, U.S.A.) to determine the protein concentration using a protein assay kit (BioRad). The eluates wer...

The N-terminal glycine-rich and cysteine-rich regions are essential for antimicrobial activity of crustinPm1 from the black tiger shrimp Penaeus monodon

“…The mouse SWAM1 and 2 have antibacterial activity but not the antiproteinase activity [11]. The WAP from rat milk inhibits the growth of S. aureus JCM2413 [13]. A related WFDC2 protein from milk of tammar wallaby also exhibits antibacterial activity against S. aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resides with the second WAP domain [30].…”

Identification and Functional Characterization of a Novel Monotreme- Specific Antibacterial Protein Expressed during Lactation

“…The role of milk in the growth, development and disease protection of young is yet to be established and there is speculation that the survival of eggs and the young of monotremes is enhanced by microbial inhibitors of cutaneous or mammary gland origin [8], [9]. Protective properties of milk in a vast range of mammals have been reported: for example, anti- parasitic activity of human milk lipase [10], antimicrobial activity of tammar wallaby milk cathelicidins and WFDC2 protein [11], [12], antifungal activity of bovine milk lactoferrin [13] and bacteriostatic activity of murine milk whey acidic protein [14]. In this study, for the first time, we identify a novel, monotreme-specific transcript that shows abundant expression in the milk cells during late- lactation.…”

A double WAP domain-containing protein PmDWD from the black tiger shrimp Penaeus monodon is involved in the controlling of proteinase activities in lymphoid organ

“…They, therefore, play a significant role in controlling the extent of neutrophil proteinase activity during inflammatory responses and tissue repair. They exhibit also the antimicrobial activity against bacteria [7,8], fungi [9,10] and viruses [11,12]. Recently, the expression of elafin and SLPI was found to be modulated in response to the onset of cancer [13e16].…”