A goose-type lysozyme gene in Japanese scallop (Mizuhopecten yessoensis): cDNA cloning, mRNA expression and promoter sequence analysis.
ABSTRACT Lysozyme is an important component of the immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. We constructed a high-quality cDNA library from mantle tissue of adult Japanese scallop (Mizuhopecten yessoensis). The EST which is high homology with g-type lysozyme genes of other species was found in the cDNA library. In the present study, the complete express sequence of g-type lysozyme genes from Japanese scallop (designated as MyLysoG) was directly obtained by PCR. The complete sequence of MyLysoG cDNA consisted of a 5' untranslated region (UTR) of 25 bp, an open reading frame (ORF) of 606 bp, and a 3' UTR of 100 bp with one polyadenylation signal (AATAAA). The deduced amino acids of MyLysoG were 201 amino acids with a putative signal peptide of 18 amino acid residues. It shared the sequence similarity and the common structure features with the g-type lysozyme from other species. Quantitative reverse trancriptase real-time PCR (qRT-PCR) assay demonstrated that mRNA transcripts of g-type lysozyme could be detected in various tissues of unchallenged scallop, and the highest expression of MyLysoG was detected in hepatopancreas tissue. The temporal expression of MyLysoG in hemolymph after Vibrio anguillarum challenge was up-regulated and reached the maximum level at 3h post stimulation, and then dropped back to the original level even lower than the control group. Furthermore, a 978 bp of 5'-flanking sequence of MyLysoG was identified by genome walking, and several potential transcription factor binding sites (TFBS) were detected in the putative promoter region. One part of the MyLysoG promoter region contains nine sites of SNPs and three sites of insert-deletion (indel) polymorphisms, and these mutations were found organize into two haplotypes. The two haplotypes were associated with different TFBS. The haplotypes could be selected to analyze the transcriptional-level control of scallop g-type lysozyme gene and the scallop immune system.
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ABSTRACT: To determine the structure and functional relationships of invertebrate lysozymes, we isolated a new invertebrate (i)-type lysozyme from the common orient clam (Meretrix lusoria) and determined the complete amino acid sequence of two isozymes that differed by one amino acid. The determined sequence showed 65% similarity to a lysozyme from Venerupis philippinarum (Tapes japonica), and it was therefore classified as an i-type lysozyme. The lytic activities of this lysozyme were similar to those of previously reported bivalve i-type lysozymes, but unlike the V. philippinarum lysozyme, it did not exhibit an increase in activity in high ionic strength. Our data suggest that this lysozyme does not have a dimeric structure, due to the replacement of Lys108 which contributes to dimer formation in the V. philippinarum lysozyme. GlcNAc oligomer activities suggested an absence of transglycosilation activity and a higher number of subsites on this enzyme compared with hen egg lysozyme.Bioscience Biotechnology and Biochemistry 11/2013; · 1.27 Impact Factor
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ABSTRACT: Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu(142) and Asp(168)) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections.Fish & Shellfish Immunology 08/2013; · 2.96 Impact Factor
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ABSTRACT: Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone , abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5 - 6.5) and over a broad temperature range (20 - 60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology.Fish & Shellfish Immunology 05/2013; · 2.96 Impact Factor