Cation ic effect

LF was found to contain an antimicrobial sequence near its N-terminus, which appears to function by a mechanism distinct from iron chelation. The identified domain contains a high proportion of basic residues, like various other antimicrobial peptides known to target microbial membranes and it appears to be located on the surface of the folded protein allowing its interaction with surface components of microbial cells (Tomita et al, 1994).

Hydrolysates prepared by cleavage of bLF with porcine pepsin, cod pepsin, or acid protease from Pénicillium duponti showed strong activity against E. coli Olll, whereas hydrolysates produced by trypsin, papain, or other neutral proteases were much less active (Tomita et al, 1991). Low molecular weight peptides generated by porcine pepsin cleavage of bLF showed broad-spectrum antibacterial activity, inhibiting the growth of a number of Gram-negative and Gram-positive species, including strains that were resistant to native LF. The antibacterial potency of the hydrolysate was at least eightfold greater than that of undigested LF with all strains tested. The active peptides retained their activity in the presence of added iron, unlike native LF. The effect of the hydrolysate was bactericidal as indicated by a rapid loss of viability of E. coli Olll.

A single active peptide representing antimicrobial domain was isolated following gastric pepsin cleavage of hLF, and bLF, and sequenced by automated Edman degradation. The antimicrobial sequence was found to consist mainly of a loop of 18 amino acid residues formed by a disulfide bond between cysteine residues 20 and 37 of hLF, or 19 and 36 of bLF (Bellamy et al, 1992). Synthetic analogs of this region similarly exhibited potent antibacterial properties. The active peptide of bLF was more potent than that of hLF having effectiveness against various Gram-negative and Gram-positive bacteria at concentrations between 0.3 |iM and 3.0 |iM, depending on the target strain. Effect of the isolated domain was lethal causing a rapid loss of colony-forming capability {Figure 6).

Human LF contains a 46 residue sequence named lactoferricin H (hLFcin) responsible for its cationic antimicrobial properties. Synthetic peptides HLT1, corresponding to the loop region of hLFcin (FQWQR-NMRKVRGPPVS) and HLT2, corresponding to its charged portion (FQWQRNMRKVR), exerted significant antibacterial effects against E. coli serotype 0111 strains NCTC 8007 and ML35 (Odell et al., 1996). The corresponding sequences in native hLF were shown to adopt a charged helix and hydrophobic tail within the N-lobe remote from the iron binding site. Sequence similarities between LFcin and dermaseptin and magainins suggest that LFcin may act as an amphipathic a-helix.

The basic amino acid-rich region of bovine lactoferricin (bLFcin), RRWQWRMKKLG has many basic and hydrophobic amino acid residues. Using chemically synthesized bLFcin and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of E. coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis (Kang et al., 1996). The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 mM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of bLFcin is involved in the interaction with bacterial phospholipid membranes and may play an important role in antimicrobial activity with little or no hemolytic activity.

To study the immunochemical and structural properties of bLFcin derived from N-lobe of bLF, monoclonal antibody (mAb) was prepared and the amino acid sequence concerned with binding to mAb identified (Shimazaki et al., 1996). Mice injected with bLFcin showed no production of antibody specific to this peptide, whereas those with bLFcin-KLH conjugate produced anti-bLFcin antibodies. None of the mAb reacted with bLF C-lobe, hLF or hLFcin. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using spots and against chemically modified derivatives of bLFcin, the antigenic determinant was identified to be the sequence 'QWR\

Furthermore, three peptides with antibacterial activity toward enterotoxigenic E. coli have been purified from a pepsin digest of bLF (Dionysius & Milne, 1997). All peptides were cationic and originated from the N-terminus of the molecule in a region where a bactericidal peptide, bLFcin, had been previously identified. The most potent peptide, peptide I, was almost identical to bLFcin; the sequence corresponded to residues 17 to 42, and the molecular mass was 3195 as determined by mass spectrometry. A second, less active peptide, peptide II, consisted of two sequences, residues 1 to 16 and 43 to 48 (molecular mass of 2673), linked by a single disulfide bond. The third peptide, peptide III, also a disulfide-linked hetero-dimer, corresponded to residues 1 to 48 (molecular mass of 5851), cleaved between residues 42 and 43. Peptides I and II displayed antibacterial activity toward a number of pathogenic and food spoilage microorganisms, and peptide I inhibited the growth of Listeria monocytogenes at concentrations as low as 2 uM. Bacterial growth curves showed that bactericidal effects of peptides I and II were observable with in 30 min of exposure. The results confirmed and extended those of earlier studies suggesting that the bactericidal domain of LF was localized in the N-terminus and did not involve iron-binding sites.

However, the antibacterial studies conducted by Hoek and co-workers (1997) indicated that the activity of LFcin is mainly, but not wholly, due to its N-terminal region. Several peptides sharing high sequence homology with bLFcin were generated from bLF with recombinant chymosin. Two peptides were co-purified, one identical to bLFcin and another differing from this cationic peptide by the inclusion of a C-terminal alanine. Two other peptides were copurified from chymosin-hydrolyzed LF, one differing from bLFcin by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond. These peptides were isolated in a single step from chymosin-hydrolyzed LF by membrane ion-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC). They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination. Pure LFcin, prepared from pepsin-hydrolyzed LF, was purified by standard chromatography techniques. This peptide was analyzed against a number of Gram-positive and Gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 l_iM or less. Sub-fragments of LFcin were isolated from reduced and cleaved peptide by reverse-phase HPLC. Sub-fragment 1 (residues 1 to 10) was active against most of the test microorganisms at concentrations of 10 to 50 |iM. Sub-fragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 ]j.M.

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