Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach.
C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.
Ibrahim
Hisham R
HR
Department of Biochemistry and Biotechnology, Faculty of Agriculture, 1-21-24 Korimoto, Kagoshima University, Kagoshima 890-0065, Japan. hishamri@chem.agri.kagoshima-u.ac.jp
Inazaki
Daisuke
D
Abdou
Adham
A
Aoki
Takayoshi
T
Kim
Mujo
M
D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects*; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D018928:Immunity, Mucosal; D013058:Mass Spectrometry; D008958:Models, Molecular; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D010434:Pepsin A / Q000378:metabolism*; D011487:Protein Conformation; D017422:Sequence Analysis, DNA
D023181:Antimicrobial Cationic Peptides; D009113:Muramidase; D010434:Pepsin A