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Identification and characterization of a novel phage-type like lysozyme from Manila clam, Ruditapes philippinarum
Jianfeng Ding1, Rui Wang1, Feng Yang1
1Dalian Ocean University, Dalian 116023, PR China; Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province, Dalian 116023, PR China.
Abstract:
A novel lysozyme gene (RpLysPh) with high similarity to the bacteriophage lysozymes was identified in Manila clam, Ruditapes philippinarum. The full length cDNA of RpLysPh is 828bp and contains a 462bp open reading frame (ORF) that codes for a 154 amino acid protein. Multiple sequence alignment analysis revealed that the three residues essential for catalytic activity in phage-type lysozyme (Glu(20), Asp(29), and Thr(35)) are conserved in RpLysPh. The comparison of the 3D models of RpLysPh and Coxiella burnetii lysozyme also suggested that the active sites involved in the binding of substrate have similar conformations. Phylogenetic analysis suggested that RpLysPh shares a similar origin with the bacterial phage-type lysozyme group. The highest level of expression of RpLysPh was observed in hemocytes, followed by mantle. Induction of RpLysPh expression was observed in gills in response to lipopolysaccharide (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)), and whole glucan particles (WGP) challenge. The recombinant protein of RpLysPh showed antibacterial activity against both Gram-positive and Gram-negative bacteria.
Insights
A novel lysozyme gene, RpLysPh, was identified in Manila clams. This gene shows antibacterial activity and is expressed in hemocytes, suggesting a role in the clam
Area of Science:
- Marine Biology
- Immunology
- Molecular Biology
Background:
- Lysozymes are crucial enzymes in innate immunity.
- Bacteriophage-type lysozymes are known for their antibacterial properties.
- The Manila clam (Ruditapes philippinarum) is an important bivalve species.
Purpose of the Study:
- To identify and characterize a novel lysozyme gene in the Manila clam.
- To investigate the expression patterns and functional activity of this lysozyme.
- To understand its potential role in the clam's immune defense.
Main Methods:
- Gene identification and sequencing (cDNA, ORF analysis).
- Multiple sequence alignment and 3D modeling for structural and functional analysis.
- Phylogenetic analysis to determine evolutionary relationships.
- Gene expression analysis (hemocytes, mantle, gills) under immune challenge (LPS, PGN, Poly(I:C), WGP).
- Recombinant protein expression and antibacterial activity assays.
Main Results:
- A novel lysozyme gene, RpLysPh, was identified in Ruditapes philippinarum.
- Key catalytic residues and active site conformation are conserved, similar to phage-type lysozymes.
- RpLysPh is primarily expressed in hemocytes and mantle, with induced expression in gills upon immune stimulation.
- Recombinant RpLysPh demonstrated broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria.
Conclusions:
- RpLysPh represents a novel bacteriophage-type lysozyme in Manila clams.
- Its conserved catalytic residues and expression pattern suggest a significant role in innate immunity.
- RpLysPh exhibits potent antibacterial activity, contributing to the clam's defense mechanisms against bacterial pathogens.
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