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Journal of Biochemistry Advance Access originally published online on October 27, 2007
Journal of Biochemistry 2008 143(1):79-86; doi:10.1093/jb/mvm195
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© 2007 The Japanese Biochemical Society.

Characterization of the {alpha}-Helix Region in Domain 3 of the Haemolytic Lectin CEL-III: Implications for Self-Oligomerization and Haemolytic Processes

Keigo Hisamatsu, Nobuaki Tsuda, Shuichiro Goda and Tomomitsu Hatakeyama*

Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan

*To whom correspondence should be addressed. Tel: +81-95-819-2686, Fax: +81-95-819-2684, E-mail: thata{at}nagasaki-u.ac.jp

Received June 2, 2007; Accepted October 7, 2007


  Abstract

CEL-III is a haemolytic lectin, which has two β-trefoil domains (domains 1 and 2) and a β-sheet-rich domain (domain 3). In domain 3 (residues 284–432), there is a hydrophobic region containing two {alpha}-helices (H8 and H9, residues 317–357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the {alpha}-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second {alpha}-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the {alpha}-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the {alpha}-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the {alpha}-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.

Key Words: antibacterial peptide, Ca2+-dependent lectin, haemolysin, oligomerization, small-angle X-ray scattering

Abbreviations: CD, circular dichroism; SAXS, small-angle X-ray scattering; GST, glutathione S-transferase; TBS, Tris-buffered saline; TSB, tryptic soy broth; PBS, phosphate-buffered saline


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