Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes.
CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-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.
Hisamatsu
Keigo
K
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan.
Tsuda
Nobuaki
N
Goda
Shuichiro
S
Hatakeyama
Tomomitsu
T
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D053878:Hemolytic Agents / Q000737:chemistry* / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D000900:Anti-Bacterial Agents; C402644:CEL-III lectin, Cucumaria echinata; D053878:Hemolytic Agents; D037102:Lectins; D010455:Peptides; D011993:Recombinant Fusion Proteins