Development of an Antibacterial MAP Homologous to a
Loop Region in Human Lactoferrin
Masachika Azuma 1
azuma@translell.eco.tut.ac.jp
Masaki Kobayashi 3

kobayashimasaki@msg.biglobe.ne.jp

Taiki Kojima 2
fwhw8642@mb.infoweb.or.jp
Carlos Adriel Del Carpio 1

carlos@translell.eco.tut.ac.jp

Laboratory for Informatics & AI in Molecular and Biological Sciences, Department ofEnvironmental Life Science, Toyohashi University of Technology, Tempaku, Toyohashi
441-8580, Japan
2 Department of Surgery II, Nagoya University, School of Medicine, Nagoya 466, Japan
3 Life Information Analysis Center Inc., Moto, Komaki 485, Japan

11 Introduction
We report on the antibacterial activities of an 11 residue peptide (FQWQRNMRKVR) homologous tojust over half the loop region of human lactoferricin. The peptide, in the form of a multiple antingen
peptide (MAP), exerted signi cant antibacterial e ects against a broad spectrum of bacteria including
MRSA.
Lactoferricin is an iron-binding glycoprotein belonging to the transferrin family and found in thespeci c granules of neutrophils and in secretion uids such as tears, saliva and even milk. The pepsin
digestion of bovine and human lactoferrin promotes the release of antibacterial peptides known as
lactoferricin B and H respectively. This may be the key to its antimicrobial activity [1]. Lactoferricin
is e ective against Gram-positive and negative bacteria as well as yeasts [2], its mechanism of action
is however unknown.
Human lactoferricin comprises two chains from residue 1-46 from the N-terminus of lactoferrin andincludes an 18 residue loop formed by one of two disulphide bridges [1]. Recently, it has been shownthat a much smaller peptide of only 11 residues (FQWQRNMRKVR), homologous to just over half
the loop region has potent antibacterial activity and may account for all the activity of the largerpeptides.
Here we describe the computer analysis of the structure of such antibacterial MAP, and the experimental results of their antibacterial activities.

2 Computer Analysis and Experimental Results
We predicted the eleven amino acid long peptide (FQWQRNMRKVR) three dimensional structureusing our 3D structure prediction program (GAX) [3]. The force eld implemented in this program
corresponds to the MM3 in vacuum. The structure of the MAP with 4 strands of the peptide wasalso predicted. However for technical reasons it was not possible to obtain the structures for higher
branching such as MAP-8 and MAP-16.
The secondary structure of FQWQRNMRKVR, derived from the predicted three dimensional
coordinates showed an -helix bordered by two turn like structures at the N and C terminals Fig. 1.
The MAP-4 structure conserved the alpha-helical structure in each strand. The structure showed thefour strands extended in a symmetrical manner, Fig. 2, although some stable conformers were also
obtained where the strands laid crossed with each other.
Experimental results of the tests of antibacterial activities show that MAP-8 and MAP-16 exhibited
anti-bacterial activity against Pseudomonas aeruginosa, while the linear MAP-2 and MAP-4 did not,up to a concentration of 200 M.
376

MAP-16, however, showed antibacterial activity against a broad spectra of bacteria, being especially strong against Pseudomonas aeruginosa.

Figure 1: -helix structure.

Figure 2: MAP4 structure for theantibacteria.

3 Conclusions
Computer modeling of the three dimensional structure of the MAP containing the 11 amino acid long(FQWQRWMRKVR) peptide strongly suggests that the structure (an amphipathic cationic alphahelix) exerts its antibacterial activity by performing ionic channels or pores in the bacterial membrane.
The results are supported by former reports that amphipathic cationic helices are common patterns
to some or the antibacterial peptides or antibacterial domains of peptides such as magainins [4],cecropins [5], and CAP18 [6].
Moreover, MAP structures were originally synthesised as stimulators of antibody production insensitized animals, and their use included vaccine development. Here we propose a novel possibility
for this type of multiple antigen peptides, i.e. the increase in the activity of anti-biotic peptides.

References[1] Bellany, W., et al., Identi cation of the bactericidal domain of lactoferrin, Biochim. Biophys.
Acta, 1121:130{136, 1992.
[2] Bellany, W., et al., Antibacterial spectrum of lactofellicinB a potent bactericidal peptide derived
from the N-terminal region of bovine lactofellin, et al., J. Appl. Bacteriol., 73:472{479, 1992.
[3] Del Carpio, C.A., A parallel genetic algorithm for polypeptide three dimensional structure prediction, A transputer implementation, J. Chem. Inf. Comp. Scie., 36(2):258{269, 1996.
[4] Matsuzaki, K., et al., Magainin 1-induced leakage of entrapped calcein out of negatively-charged
lipid vesicles, Biochim, Biochim. Biophys. Acta, 981:130{134, 1989.
[5] Fink, J., et al., The chemical synthesis of cecropin D and an analog with enhanced antibacterialactivity, J. Biol. Chem., 264:6260{6267, 1989.
[6] Tossi, A., et al., Identi cation and characterization of a primary antibacterial domain in CAP18,
a lipopolysaccharide binding protein from rabbit leukocytes, FEBS Lett., 339:108{112, 1994.
377