Characterization of a c-type lysozyme of Scophthalmus maximus: expression, activity, and antibacterial effect.
Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Lan-ping
LP
Yu
Bo-guang
BG
Sun
Jun
J
Li
Li
L
Sun
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005393:Fish Diseases / Q000276:immunology*; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005412:Flatfishes / Q000235:genetics / Q000276:immunology*; D006090:Gram-Negative Bacteria / Q000276:immunology / Q000502:physiology; D016905:Gram-Negative Bacterial Infections / Q000276:immunology / Q000662:veterinary*; D006094:Gram-Positive Bacteria / Q000276:immunology / Q000502:physiology; D016908:Gram-Positive Bacterial Infections / Q000276:immunology / Q000662:veterinary*; D015151:Immunoblotting / Q000662:veterinary; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009928:Organ Specificity; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013045:Species Specificity
D000900:Anti-Bacterial Agents; D029941:Fish Proteins; D011994:Recombinant Proteins; D009113:Muramidase