Saranya Revathy Kasthuri

A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: Genome organization, transcriptional profiling and biological activities of recombinant protein

Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function.

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 [1], 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.

Functional characterization of the evolutionarily preserved mitochondrial antiviral signaling protein (MAVS) from rock bream, Oplegnathus fasciatus.

Antimicrobial immune defense is evolutionarily preserved in all organisms. Mammals have developed robust, protein-based antiviral defenses, which are under constant investigation. Studies have provided evidences for the various fish immune factors sharing similarity with those of mammals. In this study, we have identified an ortholog of mitochondrial antiviral signaling protein from rock bream, Oplegnathus fasciatus. RbMAVS cDNA possesses an open reading frame (ORF) of 1758 bp coding for a protein of 586 amino acids with molecular mass of approximately 62 kDa and isoelectric point of 4.6. In silico analysis of RbMAVS protein revealed a caspase recruitment domain (CARD), a proline rich domain and a transmembrane domain. RbMAVS protein also contains a putative TRAF2 binding motif, (319)PVQDT(323). Primary sequence comparison of RbMAVS with other orthologues revealed heterogeneity towards the C-terminus after the CARD region. RbMAVS transcripts were evident in all the examined tissues. RbMAVS expression was induced in vivo after poly I:C challenge in peripheral blood cells, liver, head kidney and spleen tissues. Over-expression of RbMAVS potently inhibited marine birnavirus (MABV) infection in rock bream heart cells and induced various cytokines and signaling molecules in vitro. Thus, RbMAVS is an antiviral protein and potentially involved in the recognition and signaling of antiviral defense mechanism in rock bream.

Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates.

Akirin2 homologues from rock bream, Oplegnathus fasciatus: Genomic and molecular characterization and transcriptional expression analysis.

Akirins are conserved nuclear resident NF-κB signaling pathway molecules. Isoforms of akirins found in various organisms are known to play diverse roles. In this study, we have characterized two akirin2 homologues from rock bream, OfAk2(1) and OfAk2(2). The proteins derived from OfAk2(1) and OfAk2(2) revealed the presence of nuclear localization signal. Multiple sequence alignment and pairwise alignment of OfAk2(1) and OfAk2(2) with the akirin homologues, revealed high conservation and identity. Phylogenetic tree analysis revealed that the distinct position of OfAk2(1) and OfAk2(2) was close to the fish homologues and separated from the mammals and invertebrates. Genomic structure characterization revealed two distinct structures. OfAk2(1) possessed 6 exons interrupted by 5 introns whereas OfAk2(2) possessed 5 exons interrupted by 4 introns. The promoter analysis revealed the presence of significant transcription factors, which suggests its regulation by diverse stimuli. In addition, transcript expression analysis using real time quantitative reverse-transcriptase polymerase chain reaction post immune challenges with lipopolysaccharide, Edwardsiella tarda and poly I:C revealed upregulation of both OfAk2(1) and OfAk2(2) in liver, spleen and head kidney.

Characterization of MIF family proteins: MIF and DDT from rock bream, Oplegnathus fasciatus

Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule playing vital roles in various signaling cascades, including cell proliferation, and activation of immune responses against infections. It is well known as a pivotal regulator of innate immunity. In this study, we have rescued and characterized two members of the MIF family, macrophage migration inhibitory factor (OfMIF) and D-Dopachrome tautomerase (OfDDT) from rock bream, Oplegnathus fasciatus. The deduced OfMIF and OfDDT protein sequences revealed the presence of the catalytic oxidoreductase (CXXC), motif. They also possessed highly conserved proline (P(2)) and lysine residues (K(33)), responsible for their isomerase and tautomerase functions. Rock bream MIF and DDT homologues shared higher identity with fish homologues and also with mammals and occupied a distinct position in the phylogenetic tree, depicting their evolutionary conservation. The spatial expression analysis revealed the highest expression of both OfMIF and OfDDT in liver, while portraying constitutive expression in other tissues. The recombinant proteins purified using the Escherichia coli system revealed potent oxidoreductase activity against insulin with both dithiothreitol and glutathione as reducing agents. Stimulation of rock bream head kidney cells with recombinant OfMIF and OfDDT proteins induced the expression of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and interleukin-1β (IL-1β). These results together suggest their involvement in rock bream immune defense and this study on the novel MIF family member DDT from rock bream will pave the way for further studies of this homologue in other teleosts and delineate its multiple functions.

Molecular characterization and expression analysis of IκB from Haliotis discus discus

Innate immune system relies on the recognition of pathogen associated molecular patterns present in the microbes by the pattern recognition receptors leading to the activation of signaling cascade and subsequent synthesis of cytokines. NF-κB is a major stimulus activated transcription factor, which regulates the expression of a diverse array of genes. IκB is an inhibitor of NF-κB, retaining NF-κB in an inactive state in the cytoplasm. In this study, we have reported the characterization of first abalone IκB (HdIκB). The cDNA possessed an ORF of 1200 bp coding for a protein of 400 amino acids with molecular mass of 45 kDa and isoelectric point of 4.7. HdIκB protein possessed a conserved phosphorylation site (58)DSGIFS(63) in the N-terminal region, six ankyrin repeats, and a PEST sequence in the C-terminal region. A casein kinase II phosphorylation site could also be observed in the PEST sequence. Constitutive expression of HdIκB revealed its physiological significance since NF-κB is known to be activated by various stimuli. Elevated expression of HdIκB transcripts could be observed in abalones challenged with various mitogens and live microbes. This novel characterization of abalone IκB would further be a positive approach in the affirmation of evolutionary conservation and significance of this protein as a repressor/inhibitor of a pleiotropic transcription factor like NF-κB.

Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense

Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.

Genomic structural characterization and transcriptional expression analysis of proteasome activator PA28α and PA28β subunits from Oplegnathus fasciatus

Proteasomes are multicatalytic subunit complexes involved in the degradation of cytosolic proteins and antigen presentation. In this study, we have characterized the alpha and beta subunits of proteasome activator complex from rock bream at the molecular level. RbPA28α and RbPA28β possessed the characteristic features of the subunits identified from mammals and teleosts. The RbPA28α and RbPA28β proteasome subunits contained a proline-rich motif (Region A), subunit-specific insert in the region corresponding to the KEKE motif of the known PA28α (Region B), conserved activation loop (Region C), a potential protein kinase C recognition site (Region D) and a highly homologous C-terminal region (Region E) among all three PA28 subunits. Multiple sequence alignment and pairwise alignment revealed that RbPA28α and RbPA28β proteins shared high homology with the teleosts and mammals. RbPA28α and RbPA28β genome possessed 11 exons interrupted by 10 introns. In silico promoter analysis of RbPA28α and RbPA28β revealed various transcription factor-binding sites displaying their regulation under various stress conditions. Tissue distribution profiling showed a higher expression in blood and gills. Transcriptional expression analysis of RbPA28α and RbPA28β showed up-regulation in the immune tissues following LPS and poly I:C challenges, providing further evidence for the immunological role of RbPA28α and RbPA28β.