Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties

Premachandra, H.K.A.,Elvitigala, D.A.S.,Whang, I.,Kim, E.,De Zoysa, M.,Lim, B.S.,Yeo, S.Y.,Kim, S.,Park, M.A.,Park, H.C.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.34 No.6

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens

( Su Jin Lee ),( Youngdeuk Lee ),( Gun Hoo Park ),( Navaneethaiyer Umasuthan ),( Soo Jin Heo ),( Mahanama De Zoysa ),( Won Kyo Jung ),( Dae Won Lee ),( Hanjun Kim ),( Do Hyung Kang ),( Chulhong Oh ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.6

L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).

Defensin from disk abalone Haliotis discus discus: Molecular cloning, sequence characterization and immune response against bacterial infection

De Zoysa, M.,Whang, I.,Lee, Y.,Lee, S.,Lee, J.S.,Lee, J. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.28 No.2

Gene-encoded antimicrobial peptides (AMPs) serve a major role in host defense systems against pathogens. In this study, cDNA of a new mollusk defensin was identified from a normalized cDNA library constructed from whole tissues of disk abalone. Abalone defensin peptide (pro-defensin) has a 198-bp coding sequence comprised of a putative 66 amino acids with a mature defensin consisting of 48 amino acid residues. The presence of an invertebrate defensin family domain, an arrangement of six cysteine residues and their disulfide linkage in C<SUB>1</SUB>-C<SUB>4</SUB>, C<SUB>2</SUB>-C<SUB>5</SUB> and C<SUB>3</SUB>-C<SUB>6</SUB> form, an alpha helix in three-dimensional structure and a phylogenetic relationship suggests that abalone defensin could be a new member of the invertebrate defensin family, and related to arthropod defensins. In non-stimulated abalone, defensin transcripts were constitutively expressed in all examined tissues including hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas. It was observed that abalone defensin transcripts were significantly induced in hemocytes, gills and digestive tract at different time intervals after infection by pathogenic bacteria mixture containing Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes. Our overall results suggest that disk abalone defensin could be involved in the immune response reactions as a host defense against pathogenic bacteria.

First molluscan transcription factor activator protein-1 (Ap-1) member from disk abalone and its expression profiling against immune challenge and tissue injury

De Zoysa, Mahanama,Nikapitiya, Chamilani,Lee, Youngdeuk,Lee, Sukkyoung,Oh, Chulhong,Whang, Ilson,Yeo, Sang-Yeop,Choi, Cheol Young,Lee, Jehee Elsevier 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.6

<P><B>Abstract</B></P><P>The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone <I>Haliotis discus discus</I> (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (<I>Takifugu rubripes</I>), human and insect (<I>Ixodes scapularis</I>) Ap-1, respectively.</P><P>Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (<I>Vibrio alginolyticus, Vibrio parahemolyticus</I> and <I>Lysteria monocytogenes</I>) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.</P>

Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H₂O₂ oxidative stress and Vibrio alginolyticus bacterial infection

De Zoysa, M.,Pushpamali, W.A.,Oh, C.,Whang, I.,Kim, S.J.,Lee, J. Academic Press 2008 FISH AND SHELLFISH IMMUNOLOGY Vol.25 No.4

Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at <SUP>223</SUP>TGA<SUP>225</SUP> that corresponds to selenocysteine (Sec) amino acid as U<SUB>75</SUB>. The full length cDNA consists of 675bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 (<SUP>97</SUP>LGFPCNQF<SUP>104</SUP>), an active site motif (<SUP>183</SUP>WNFEKF<SUP>188</SUP>) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3' UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H<SUB>2</SUB>O<SUB>2</SUB> injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H<SUB>2</SUB>O<SUB>2</SUB> oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H<SUB>2</SUB>O<SUB>2</SUB> oxidative stress and immune defence against bacterial infection.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-α factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus)

De Zoysa, M.,Nikapitiya, C.,Oh, C.,Whang, I.,Lee, J.S.,Jung, S.J.,Choi, C.Y.,Lee, J. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.28 No.5-6

The lipopolysaccharide-induced TNF-α factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn<SUP>+2</SUP> binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-α expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-α, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca.

First report of invertebrate Mx: Cloning, characterization and expression analysis of Mx cDNA in disk abalone (<i>Haliotis discus discus</i>)

De Zoysa, Mahanama,Kang, Hyun-Sil,Song, Young-Bo,Jee, Youngheun,Lee, Young-Don,Lee, Jehee Elsevier 2007 FISH AND SHELLFISH IMMUNOLOGY Vol.23 No.1

<P><B>Abstract</B></P><P>Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN α/β) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (<I>Haliotis discus discus</I>) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5′-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L<SUB>468</SUB>, L<SUB>475</SUB>, L<SUB>489</SUB> and L<SUB>510</SUB>, suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.</P>

Abhisin: A potential antimicrobial peptide derived from histone H2A of disk abalone (<i>Haliotis discus discus</i>)

De Zoysa, Mahanama,Nikapitiya, Chamilani,Whang, Ilson,Lee, Jae-Seong,Lee, Jehee Elsevier 2009 FISH AND SHELLFISH IMMUNOLOGY Vol.27 No.5

<P><B>Abstract</B></P><P>Antimicrobial peptides (AMPs) play an important role in the immune defense against pathogenic microorganisms. In this study, a histone H2A full-length cDNA was cloned from disk abalone <I>Haliotis discus discus</I>. We identified a 40-amino acid AMP designated as abhisin from the N-terminus of the abalone histone H2A sequence. Abhisin shows the characteristic features of AMPs including net positive charge (+13), higher hydrophobic residues (27%) and 2.82 kcal/mol protein binding potential. Abhisin shares 80% amino acid identity with the buforin I peptide that is derived from Asian toad histone H2A. We synthesized the synthetic peptide of abhisin, and characterized its antimicrobial activities. Our results showed the growth inhibition of Gram positive (G+) <I>Listeria monocytogenes</I>, Gram negative (G-) <I>Vibrio ichthyoenteri</I> bacteria, and fungi (yeast) <I>Pityrosporum ovale</I> by abhisin treatment at 250 μg/mL. However, stronger activity was displayed against the G+ than G− bacteria. Additionally, scanning electron microscope (SEM) observation results confirmed that <I>P. ovale</I> cells were damaged by abhisin treatment. Interestingly, abhisin treatment (50 μg/mL) decreased the viability of THP-1 leukemia cancer cells approximately by 25% but there was no effect on the normal vero cells, suggesting that abhisin has cytotoxicity against cancer cells but not normal cells. Quantitative real time RT-PCR results revealed that histone H2A transcription was significantly induced at 3 h post-infection with bacteria in abalone gills and digestive tract. These results suggest that abhisin is a potential antimicrobial agent, and its precursor histone H2A may be involved in the innate immune defense system in abalone.</P>

Collective Forest Management System in Japan: a Case Study in Osawa Property Ward Forest

De Zoysa, Mangala Premakumara,Inoue, Makoto,Yamashita, Utako,Hironori, Okuda Institute of Forest Science 2013 Journal of Forest Science Vol.29 No.1

Iriai an Indigenous forest management system in Japan from the viewpoint of "common pool resources" was a success resilient institution and resulted with sustainable production system and environmental conservation. This study was conducted in Osawa of the Nagano prefecture through group discussions, field observations and an in-depth field survey. Osawa Property Ward Forest is managed under the concept very much similarly to traditional "Iriai". This study firstly examined the changes of collective forest management system in terms of awareness and interest in forest management; forest management activities; role of forest; and collection of forest products. Then it analyzed the current threats for collective forest management have been identified as: land abandonment due to loss of benefits and lack of active community participation; deterioration of forest environment particularly the micro-climate and aesthetic values; conflict with local government authorities restraining the use of money in property ward forest and conflict with outsiders on damping of the garbage. Community cantered forestry management rules; livelihood contribution; protection of environment; local initiatives for protection and economic activities are the prevailing opportunities for collective forest management. The main requirements for revitalization of collective forest management are explained as local reciprocity; imposition of community based forest rules; encouraging local innovations; and building partnerships with stakeholders. Collective forest management system addresses the limitations of conventional forestry models, which had invalidated traditional 'iriai' institutions, and key to restoring sustainable use of forest and environmental resources. Cross-institutional collaborations together with responsibilities of local communities would ensure the revitalization of forest resources.

Microarray analysis of gene expression in disk abalone Haliotis discus discus after bacterial challenge

De Zoysa, M.,Nikapitiya, C.,Oh, C.,Lee, Y.,Whang, I.,Lee, J.S.,Choi, C.Y.,Lee, J. Academic Press 2011 Fish & shellfish immunology Vol.30 No.2

In this study, we investigated the gene expression profiling of disk abalone, Haliotis discus discus challenged by a mixture of three pathogenic bacteria Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes using a cDNA microarray. Upon bacteria challenge, 68 (1.6%) and 112 (2.7%) gene transcripts changed their expression levels >=2 or @?2 -fold in gills and digestive tract, respectively. There were 46 tissue-specific transcripts that up-regulated specifically in the digestive tract. In contrast, only 13 transcripts showed gill-specific up-regulation. Quantitative real-time PCR was performed to verify microarray data and results revealed that candidate genes namely Kruppell-like factor (KLF), lachesin, muscle lim protein, thioredoxin-2 (TRx-2), nuclear factor interleukin 3 (NFIL-3) and abalone protein 38 were up-regulated. Also, our results further indicated that bacteria challenge may activate the transcription factors or their activators (Kruppell-like factor, inhibitor of NF-κB or Ik-B), inflammatory cytokines (IL-3 regulated protein, allograft inflammatory factor), other cytokines (IFN-44-like protein, SOCS-2), antioxidant enzymes (glutathione-S-transferase, thioredoxin-2 and thioredoxin peroxidase), and apoptosis-related proteins (TNF-α, archeron) in abalone. The identification of immune and stress response genes and their expression profiles in this microarray will permit detailed investigation of the stress and immune responses of abalone genes.