Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus)

Premachandra, H.K.A.,Wan, Q.,Elvitigala, D.A.S.,De Zoysa, M.,Choi, C.Y.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.38 No.4

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.

Identification of a novel molluscan short-type peptidoglycan recognition protein in disk abalone (Haliotis discus discus) involved in host antibacterial defense

Premachandra, H.K.A.,Elvitigala, D.A.S.,Whang, I.,Lee, J. Academic Press 2014 Fish & shellfish immunology Vol.39 No.1

Peptidoglycan recognition proteins (PGRPs) are a widely studied group of pattern recognition receptors found in invertebrate as well as vertebrate lineages, and are involved in bacterial pathogen sensing. However, in addition to this principal role, they can also function in multiple host defense processes, including cell phagocytosis and hydrolysis of peptidoglycans (PGNs). In this study, a novel invertebrate short-type PGRP was identified in disk abalone (Haliotis discus discus) designated as AbPGRP. The complete coding sequence of AbPGRP was 534 bp, encoding a 178-amino acid protein with a predicted molecular mass of 20 kDa. The AbPGRP gene had a bipartite arrangement consisting of two exons separated by a single intron. Homology analysis revealed that AbPGRP shares conserved features, including amino acid residues critical for substrate and ion binding as well as for its amidase activity, with homologs of other species. Phylogenetic analysis of AbPGRP revealed that it likely evolved from a common ancestor of invertebrates, having significant homology with other molluscan PGRPs. Recombinant AbPGRP exhibited detectable, dose-dependent PGN-hydrolyzing activity with the presence of Zn<SUP>2+</SUP>, and strong antibacterial activity against Vibrio tapetis, consistent with the functional properties previously reported for PGRPs in other mollusks. Moreover, AbPGRP transcription was induced upon treatment of healthy abalones with bacterial peptidoglycan and lipopolysaccharide, although the expression profiles differed with treatment, suggesting a capacity for discriminating between bacterial pathogens through molecular pattern recognition. Collectively, the findings of this study indicate that AbPGRP is a true homolog of invertebrate PGRPs and likely plays an indispensable role in host immunity.

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.

LIBERALIZATION OF THE DOMESTIC FINANCIAL MARKET

ATHUKORALA, PREMACHANDRA,RAJAPATIRANA, SARATH 한국국제경제학회 1993 International Economic Journal Vol.7 No.4

The failure of financial liberalization reforms in several developing countries in recent years has led to a reassessment of the McKinnon-Show financial liberalization paradigm. The purpose of this paper is to delineate the key issues of the debate and to throw some light on them by systematically tracing out the Sri Lankan financial policy reforms initiated in 1977. Our results challenge the neo-structuralist proposition that financial liberalization is merely a theoretical aberration unrelated to the objective conditions of developing countries and suggest that the lackluster outcome of reforms emanates mostly from their half-hearted nature, and policy inconsistencies and mistakes that crop up in the implementation process. [G18]

Edge-Based Tracking of an LED Traffic Light for a Road-to-Vehicle Visible Light Communication System

H. Chinthaka N. Premachandra,Tomohiro Yendo,Mehrdad Panahpour Tehrani,Takaya Yamazato,Toshiaki Fujii,Masayuki Tanimoto,Yoshikatsu Kimura 한국방송·미디어공학회 2009 방송공학회논문지 Vol.14 No.4

Marine teleost ortholog of catalase from rock bream (Oplegnathus fasciatus): Molecular perspectives from genomic organization to enzymatic behavior with respect to its potent antioxidant properties

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Priyathilaka, T.T.,Kim, E.,Lim, B.S.,Jung, H.B.,Yeo, S.Y.,Park, H.C.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.35 No.4

Catalases are well known antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3303 bp) of RbCat is comprised of a 1581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor-binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important amino acids present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity.

A teleostean counterpart of ferritin M subunit from rock bream (Oplegnathus fasciatus): An active constituent in iron chelation and DNA protection against oxidative damage, with a modulated expression upon pathogen stress

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Oh, M.J.,Jung, S.J.,Park, C.J.,Lee, J. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.35 No.5

Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin ''M'' which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H<SUB>2</SUB>O<SUB>2</SUB>-mediated DNA damage.

Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Nam, B.H.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.35 No.2

Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense.

Molecular cloning, expression and functional characterization of a teleostan cytokine-induced apoptosis inhibitor from rock bream (Oplegnathus fasciatus)

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Yeo, S.Y.,Choi, C.Y.,Noh, J.K.,Lee, J. Pergamon Press ; Elsevier Science 2015 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.52 No.1

Apoptosis plays a key role in the physiology of multicellular organisms and is regulated by different promoting and inhibitory mechanisms. Cytokine-induced apoptotic inhibitor (CIAPI) was recently identified as a key factor involved in apoptosis inhibition in higher vertebrate lineages. However, most of the CIAPIs of lower vertebrate species are yet to be characterized. Herein, we molecularly characterized a teleostan counterpart of CIAPI from rock bream (Oplegnathus fasciatus), designating as RbCIAPI. The complete coding region of RbCIAPI was consisted of 942 nucleotides encoding a protein of 313 amino acids with a predicted molecular mass of ~33@?kDa. RbCIAPI gene exhibited a multi-exonic architecture, consisting 9 exons interrupted by 8 introns. Protein sequence analysis revealed that RbCIAPI shares significant homology with known CIAPI counterparts, and phylogenetic reconstruction confirmed its closer evolutionary relationship with its fish counterparts. Ubiquitous spatial distribution of RbCIAPI was detected in our quantitative real time polymerase chain reaction (qPCR) analysis, where more prominent expression levels were observed in the blood and liver tissues. Moreover, the RbCIAPI basal transcription level was found to be modulated by different bacterial and viral stimuli, which could be plausibly supported by our previous observations on the transcriptional modulation of the caspase 3 counterpart of rock bream (Rbcasp3) in response to the same stimuli. In addition, our in vitro functional assay demonstrated that recombinant RbCIAPI could detectably inhibit the proteolysis activity of recombinant Rbcasp3. Collectively, our preliminary results suggest that RbCIAPI may play an anti-apoptotic role in rock bream physiology, likely by inhibiting the caspase-dependent apoptosis pathway. Therefore, RbCIAPI potentially plays an important role in host immunity by regulating the apoptosis process under pathogenic stress.

Molecular profile and expressional modulation of a Toll like receptor-1 homolog from rock bream (Oplegnathus fasciatus)

Elvitigala, Don Anushka Sandaruwan,Premachandra, H. K. A.,Yeo, Sang-Yeob,Choi, Cheol Young,Whang, Ilson,Lee, Jehee Springer-Verlag 2015 Genes & Genomics Vol.37 No.5