Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus.
Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.
Kasai
Kosuke
K
Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Japan.
Ishikawa
Takashi
T
Komata
Takafumi
T
Fukuchi
Kaori
K
Chiba
Mitsuru
M
Nozaka
Hiroyuki
H
Nakamura
Toshiya
T
Sato
Tatsusuke
T
Miura
Tomisato
T
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004817:Epidermis / Q000201:enzymology; D029941:Fish Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D005432:Flounder / Q000235:genetics / Q000378:metabolism*; D005880:Gills / Q000201:enzymology; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D007150:Immunohistochemistry; D007526:Isoelectric Point; D050830:L-Amino Acid Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009093:Mucus / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013696:Temperature
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D018076:DNA, Complementary; D029941:Fish Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050830:L-Amino Acid Oxidase