Identification and characterization of l-amino acid oxidase 2 gene in orange-spotted grouper (Epinephelus coioides)

Jiann-Horng Leu; Chi-Hang Tsai; Chia-Hsun Yang; Hsin-Yiu Chou; Hao-Ching Wang

doi:10.1016/j.dci.2021.104058

Identification and characterization of l-amino acid oxidase 2 gene in orange-spotted grouper (Epinephelus coioides)

Dev Comp Immunol. 2021 Jul:120:104058. doi: 10.1016/j.dci.2021.104058. Epub 2021 Feb 28.

Authors

Jiann-Horng Leu¹, Chi-Hang Tsai², Chia-Hsun Yang², Hsin-Yiu Chou³, Hao-Ching Wang⁴

Affiliations

¹ Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, ROC; Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan, ROC. Electronic address: jiholeu@ntou.edu.tw.
² Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, ROC.
³ Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan, ROC; Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, ROC.
⁴ The PhD Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 115, Taiwan, ROC; Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan, ROC; International Center for the Scientific Development of Shrimp Aquaculture, National Cheng Kung University, Tainan, Taiwan, ROC. Electronic address: wanghc@tmu.edu.tw.

PMID: 33657430
DOI: 10.1016/j.dci.2021.104058

Abstract

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

Keywords: Antimicrobial activity; Epinephelus coioides; Protein model; l-amino acid Oxidase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Bass / genetics
Bass / immunology*
Bass / microbiology
Cloning, Molecular
Fish Diseases / immunology*
Fish Proteins / genetics
Fish Proteins / isolation & purification
Fish Proteins / metabolism*
L-Amino Acid Oxidase / genetics
L-Amino Acid Oxidase / isolation & purification
L-Amino Acid Oxidase / metabolism*
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification
Recombinant Proteins / metabolism
Sequence Alignment
Sf9 Cells
Spodoptera
Substrate Specificity / immunology
Vibrio parahaemolyticus / immunology

Substances

Fish Proteins
Recombinant Proteins
L-Amino Acid Oxidase