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Transcriptome wide discovery of selected role players in acute phase response of black rockfish (Sebastes schlegelii) and antioxidative defense of rock bream (Oplegnathus fasciatus), revealing their putative significance in host immune responses

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Abstract
돌돔 (Oplegnathus fasciatus)과 조피볼락 (Sebastes schlegeli)은 특히 한국과 일본 같은 아시아 태평양 지역의 대부분의 국가에서 매우 수요가 높은 어류이다. 하지만, 돌돔과 조피볼락 수산양식산업에서 다양한 세균, 바이러스 또는 기생충 더불어 다른 스트레스 요인들에 의한 병원체 감염의 유행은 상당한 폐사량을 야기하고 있고 이에 따라 적절한 질병 관리 전략의 필요성이 촉구되고 있다. 그래서, 이 어종에 대한 지속 가능한 수산양식을 보증할 수 있는 적절한 질병 또는 스트레스에 저항하는 기술을 개발하기 위해서 면역 방어와 산화 방어 기전에 대한 조사가 중요하다.
이 연구의 첫 장 (chapter 1)에서 우리는 조피볼락으로부터 C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp)로 불리는 3개의 acute phase proteins (APPs)를 동정하고 분자적 특성을 분석하였으며 병원체 스트레스 상태에서 이 유전자들의 전사적 조절을 판독하였다. C-reactive protein은 pentraxin superfamily proteins에 속하며 동물에서 pattern recognition receptor molecule과 major positive APP로써 그 기능이 알려져 있다. 첫번째 연구에서 CRP-like molecule의 cDNA 서열을 이전에 구축한 조피볼락 cDNA database (RfCRP)로부터 동정하였고 그 뒤에 분자적 수준에서 특성을 분석하였다. RfCRP의 전체 암호화 서열은 672bp이고 예상 분자량이 25.19 kD인 224아미노산을 암호화하고 있었다. 아미노산서열 분석에서 RfCRP는pentraxin family signature를 포함한 pentraxin family member의 전형적인 특징이 확인되었다. Multiple sequence alignment 결과는 RfCRP가 기능적으로 중요한 잔기가 보존된 것을 보여주었다. 다른 분류군으로부터 다른 pentraxin counterparts를 사용하여 계통수를 재구축한 것에 따르면, RfCRP는 일반적인 척추동물의 조상 기원을 공유하였고 대부분의 해양 어류의 CRP와 군을 이루었다. 뿐만 아니라, 재조합 RfCRP는 Escherichia coli에 대응하여 Ca2+-의존적 응집 활성을 보여주었는데 이것은 탄수화물을 기초로 한 ligand의 존재를 완벽히 억제하였다. 게다가, 재조합 RfCRP는 또한 E. coli와 Streptococcus iniae에 대하여 항균활성을 보여주었다. 또한, qPCR분석은 RfCRP가 생리적으로 중요한 조직에서 모두에서 발현하였으며 비장에서 확연하게 발현하였다. 건강한 어류에 polysaccharides 또는 살아있는 S. iniae를 처리한 후에 RfCRP의 발현은 비장과 두신조직에서 상당히 상향조절되었다. 종합하자면, 우리의 결과는 RfCRP가 숙주의 항균방어에 중요하고 감염의 급성기에 참여하는 것으로 여겨진다.
우리의 두 번째 연구인 APPs는 조피볼락으로부터 동정된 SAA counterpart 에 특히 패혈증상에서 발현 행동에 관하여 실질적 통찰을 얻는데 집중하였다. Serum amyloid A (SAA)는 감염이나 염증의 급성기에 관련된 중요한 단백질이다. SAAs는 자기 파괴 메커니즘이나 병원균에 의해서 손상되는 건강한 세포를 보호하기 위하여 염증상태에서 간세포로부터 방출된다. 이 연구에서 이전에 구축한 조피볼락 cDNA library는 SAA homolog (RfSAA)의 전체 cDNA 서열을 동정하고 분자적 수준에서 특성을 분석하는데 사용되었다. 예상대로, in silico analysis에서 이 homolog는 이전에 이 유전자의 알려진 counterparts의 전형적인 도메인을 보여주었다. RfSAA 의 Open reading frame 은 313bp의 DNA 서열로 구성되었다. RfSAA의 파생된 폴리펩타이드는 121개의 아미노산서열을 가지고 있었으며 분자량은 13kD이다. 계통수 분석과 pairwise sequence alignment의 결과는 RfSAA가 Epinephelus coioides ortholog와 가깝게 관련되어 있음을 보여주었다. 비록 RfSAA가 분석한 모든 조직에서 발현되었지만, 간 조직에서 탁월하게 발현되었다. Quantitative real-time PCR 분석에서는 RfSAA가 간조직에서 세균성과 바이러스성 자극에 의해서 상당히 상향 조절되었으며 이것은 1차 숙주 면역 방어선의 급성기에서 중요성을 확인할 수 있다.
세 번째 연구에서, 우리는 조피볼락으로부터 Hp counterpart를 동정하고 병원체 스트레스 상태에서 이 유전자의 발현 조절에 집중하여 분자적으로 특성을 분석하였다. Haptoglobin (Hp)는 간에서 합성되는 양성 급성 단백질이며, 감염의 급성기 동안 농도가 증가되었다. Hp는 산화 손상을 포함한 자유 hemoglobin에 의한 부정적인 효과를 막기위해서 용혈과정동안 hemoglobin (Hb)에 결합한다. 또한 여기에서 우리는 이전에 구축한 조피볼락의 cDNA library는Hp homolog (RfHp)의 전체 cDNA 서열을 동정하기 위하여 사용하였고 분자적 수준에서 특성을 분석하였다. 예상대로, in silico analysis에서 RfHp는 이전에 알려진 counterpart의 전형적인 도메인을 포함하는 것을 보여주었다. RfHp의 Open reading frame은 942bp DNA 서열로 이루어져있었다. RfHp의 파생된 아미노산 서열은 313 아미노산으로 구성되어 있었으며 예상되는 분자량은 34 kD이다. 계통수 분석과 as pairwise sequence alignment의 결과는 RfHp가 진화적인 관점에서 Oreochromis mossambicus의 counterpart에 가장 가깝게 관련되어 있었다. 비록 RfHp가 실험한 모든 조직에서 발현되었지만, 간조직에서 특히 발현되었으며 이 유전자의 기원이 간세포임을 시사한다. Quantitative real-time PCR 분석에서는 RfHp 가 간조직에서 세균성과 바이러스성 자극에 의해서 상당히 상향 조절되었으며 이것은 1차 숙주 면역 방어선의 급성기에서 중요성을 확인할 수 있다.
이 연구의 두 번째 장 (chapter 2)에서 우리는 돌돔으로부터 4개의 분별되는 항산화제를 동정과 특성 분석을 시도하였다. 이 중 2개(catalase 와 thioredoxin reductase 3 (TrxR3))는 항산화 방어에 직접적으로 관여하는 것으로 알려져 있고, 다른 두 개 (ferritin H and M)는 'Fenton' 유형의 반응과 같은 산화반응의 촉매(Fe2+)를 박탈하여 항산화 방어에 간접적으로 참여한다.
두 번째 장의 첫 번째 연구는 돌돔(Oplegnathus fasciatus)으로부터 catalase counterpart동정하고 특성을 분석하였다. Catalases는 산화스트레스를 방어하기 위하여 주로 과산화수소를 물과 산소로 불균등화 반응을 하는 항산화 효소로 알려져 있다. 돌돔 (Oplegnathus fasciatus)의 Catalase의 전체 genomic DNA (gDNA) 서열은 our custom-constructed BAC genomic DNA library으로부터 동정되었고, RbCat로 명시하였다. RbCat는 12개의 intron에 의하여 분리된 13개의 exon으로 13,722bp gDNA서열로 이루어져있다. RbCat의 전체 cDNA 서열(3,303bp)은 이론상의 등전점이 8.34이고 분자량이 60 kD인 길이가 527개의 아미노산으로 된 펩타이드를 암호화한 1,581bp의 코드영억으로 구성되었다. RbCat의 참여된 프로모터 지역은 면역과 항산화 반응의 신호전달 분자에 결합되는 부위를 포함하여 여러 전자 인자 결합 부위를 포함하고 있으며 이것은 실질적인 전사 조절을 시사하고 있다. RbCat는 전형적인 catalase family signature와 유사하였고 즉, catalase proximal heme-ligand signature motif와 마찬가지로 catalase proximal active site motif로 구성되었으며 다른 어류들과 높은 상동성을 공유하였다. multiple sequence alignment에 따르면, RbCat 에서 기능적으로 중요한 아미노산은 척추동물 사이에 완벽하게 보존되어 있었다. 계통수 분석에서는 RbCat는 척추동물의 기원으로부터 진화되었고 그리고 더 나아가 어류의 분기군에 위치해 있었다. 재조합 RbCat는 기질인 과산화수소에 대하여 농도-의존적 방법에 의하여 분명한 과산화 효소 활성을 가지고 있었다. 하지만, 이것은pH와 온도의 넓은 범위 내에서 상당한 과산화 효소 활성을 보여주었다. 조직에 따라서 각각 다른 양의 RbCat mRNA 발현을 확인할 수 있었고, 이것은 조직 종류에 따라서 생리적인 다양한 역할을 시사한다. 게다가, 살아있는 병원체인Edwardsiella tarda, rock bream iridovirus (RBIV)와 유사체인 polyinosinic:polycytidylic acid, lipopolysaccharide 를 사용하여 면역 공격 실험하였을 때 면역 자극에 의하여 RbCat의 전사가 조절되었다. 종합하자면, 이 연구에서 얻은 결과들은 RbCat가 돌돔에서 강한 항산화 효소로서 기능을 하고, 과산화 효소 활성에 따라 면역 반응 후기에서 역할을 할 것이다.
이 장의 두 번째 연구는 돌돔의TrxR3 counterpart 분자적인 특성을 분석하는데 전념하였다. Thioredoxin (Trx) 시스템은 활성산소종의 과잉을 불균등화반응에 의해 세포의 항산화 방어에 중요한 역할을 한다. 그래서, Trx enzyme cascade의 개시에 관여하기 때문에 thioredoxin reductase (TrxR)의 역할을 무시할 수 없었다. 여기에, 우리는 다른 척추동물의 TrxR-3 isoforms과 높은 유사성을 보여주는 teleostean TrxR (RbTrxR-3) ortholog의 동정과 분자적 특성에 대하여 보고하고자 한다. 완전한 RbTrxR-3 암호화 서열은 예상 분자량이 66 kD이상 되는 600 아미노산을 암호화하고 있는 1800 뉴클레오티드로 구성된다. RbTrxR-3은 총 길이가 12658bp가 되는 15개의 intron에 의해 나누어진 16개의 exon으로 구성되어 있다. RbTrxR-3의 단백질 서열의 In silico analysis에서는 전형적인 typical TrxR domain architecture를 지니는 것으로 나타났다. 게다가, multiple sequence alignment 와pairwise sequence alignment strategies를 사용하였을 때, 우리는 RbTrxR-3가 다른 어류의 TrxR-3 단백질과 매우 보존된 활성 부위 잔기를 포함하여 전체적으로 높은 서열 유사성을 가지고 있는 것을 보여주었다. RbTrxR-3의 계통수에서는 어류의 TrxR-3 orthologs와 같은 군으로 나타남으로써 가까운 진화적인 관계를 알 수 있었다. RbTrxR-3 전사적 분석은 quantitative polymerase chain reaction (qPCR)을 사용하여 실시하였고 RbTrxR-3가 모든 조직에서 발현되고 가장 높게 발현하는 조직은 혈액이고 그 다음으로 아가미, 간이다. 살아있는 병원균과 바이러스 자극은 같은 병원성 스트레스에서 돌돔의 thioredoxin1과 일시적으로 연관되어있는 간에서 RbTrxR-3의 기본적인 전사의 변동을 촉발시켰다. 마지막으로, TrxR 단백질의 전형적인 기능부위를 재조합하고, 정제된 재조합 RbTrxR-3가 5,5′-dithiobis (2-nitrobenzoic) acid에 대하여 농도-의존적으로 반응하는 것을 확인할 수 있었다. 종합하자면, 이 결과들은 RbTrxR-3가 산화와 병원성 스트레스에서 숙주의 Trx 시스템에서 역할을 맡고 있는 것을 시사하고 있다.
세 번째 연구로 우리는 ferritin complex를 구성하는 중요한 요소로써 돌돔의 FerH like subunit을 동정하였다. Ferritins은 세포의 환결에서 물질대사나 철의 항상성 유지에 참여하는 24 subunit로 만들어진 철 결합 단백질이다. 여기에서, 우리는 rock bream (Oplegnathus fasciatus; RbFerH) 으로부터 ferritin의 subunits 중에 한 가지 타입(ferritin H-like subunit)의 특성을 분석하였다. RbFerH의 완전한 암호화 서열은 예상되는 분자량이 20.8 kD의 177 아미노산을 암호화하고 있는 길이가 531bp인 서열이다. 추론되는 단백질 구조는 철 결합을 위한 metal ligand, ferroxidase center와 두 개의 two iron-binding region signature를 포함한 알려진 ferritin H subunit의 특징적인 domain architecture를 가지고 있었다. 예상대로 RbFerH cDNA 서열의 5′ 비해석부위에는 전사에 관여하는 특징적인 조절 요소로 철 반응 요소 부위를 포함하고 있었다. RbFerH유전자는 4195bp로 구성된 4 개의 intron과 5 개의 exon으로 구성되어 있다. 과발현된 재조합 RbFerH 단백질은 산화된 이중나선 DNA 피해에 대응하여 보호하는 효과와 세균 발육 억제 특성, 현저한 Fe(II) 이온 박탈 활성을 보여주었다. quantitative polymerase chain reaction (qPCR) 사용하여, 우리는 돌돔에서 생리적으로 중요한 조직에서 모두 발현되는 것을 확인하였다. 높은 mRNA 전사는 혈액과 간 조직에서 확인되었다.
다른 미생물 병원체와 병원체에서 유래한 유사분열물질을 주입하였을 경우, RbFerH 전사는 현저하게 돌돔의 혈액에서 증가한다. 종합하자면, 우리가 찾아낸 것은 RbFerH가 돌돔에서 강한 iron sequestrator로서 역할을 하고, 항균성과 항산화 방어에 관여하여 활발히 관여할 것으로 생각된다.
두 번째 장의 마지막 연구로 우리는 돌돔으로부터 FerM like subunit의 특성을 분석하였다. Ferritin "M" subunit는 또한 H and L subunit 둘 다에서 특징적인 특성을 지니는 ferritin nano-cage (24 mer)를 형성하느데 기여하고 대부분의 어류는 하등 척추동물로부터 최근에 동정 된다. 이 연구에서, 돌돔 (Oplegnathus fasciatus)의 ferritin M-like subunit(RbFerM)은 분자적 수준에서 특성을 분석하였고 그리고 건강한 어류와 병원체와 유사체 자극을 받은 어류에서 이 전사적 분석을 실시하였다. 게다가. 이 유전자의 기능적인 특성을 재조합 단백질을 사용하여 평가하였다. RbFerM의 완전한 암호화 서열은 계산된 분자량이 20 kD인 176개의 아미노산을 암호화하고있는 길이가 528bp인 서열이다. RbFerM의 In silico analysis에서 다른 포유류의 ferritin subunits H와 L의 비슷한 특징을 가지고 있는 것으로 나타났다. 계통수 분석은 다른 어류들과 RbFerM이 매우 진화적으로 가까운 것으로 확인되었다. Quantitative real time polymerase chain reaction (PCR) 분석은 돌돔의 선택된 조직에서 RbFerM이 전자적으로 모두 발현되었고 혈액과 간 조직에서 좀 더 확연한 발현을 관찰할 수 있었다. 시간 경과에 따른 면역 공격 실험에서 lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, rock bream irido virus (RBIV)에 노출되었을 때 간 조직에서 상당한RbFerM의 전자적 유도를 확인할 수 있었다. RbFerM 의 정제된 재조합 단백질은 온도에 의하여 변하는 철 결합 활성을 증명하였다. 게다가 재조합 RbFerM은 iron (II)과 H2O2-mediated DNA damage에 대응하여 확연한 보호 효과를 보여주었다.
종합하자면, 이 연구의 궁극적인 목적은 돌돔과 조피볼락에서 선택된 APP counterparts와 antioxidants를 각각 밝히고 더 나아가 병원체 스트레스에 반응하는 전사적 수준에서 발현의 조정을 판독하고자 한다. 게다가, 몇몇의 분자는 면역이나 어류 생리에서 기능적인 역할을 제시된 in-vitro에서의 예상되는 기능을 보여주었다. 이런 우리의 공동연구의 결과는 병원체 감염이나 항산화 스트레스에 대응하여 어류의 생존을 높일 수 있는 점진적인 전략을 향상하는데 도움을 줄 것이다.
Rock bream (Oplegnathus fasciatus) and black rockfish (Sebastes schlegeli) are highly demanded fish delicacies in most of the countries in Asia Pacific region, especially in Korea and Japan. However, prevalence of pathogenic infections caused by various bacterial, viral or parasitic agents along with other stress factors instigates considerable mortalities in rock bream and rockfish aquaculture industries, urging the need of proper disease management strategy. Thus, it is important to investigate on naturally existing host immune defense and oxidative defense mechanisms in those aqua-crops to develop proper disease or stress resistance stretergies, assuring the sustainable aquaculture of these fish species.
In the first phase (chapter 1) of the studies we sought to identify and molecularly characterize three acute phase proteins (APPs), namely C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hp) from black rockfish, deciphering their transcriptional modulation under induced pathogen stress. C-reactive protein is categorized under pentraxin superfamily proteins, and known to function as a pattern recognition receptor molecule and major positive APP in animals. In our first study, a cDNA sequence of a CRP-like molecule was identified from a previously constructed black rockfish cDNA database (RfCRP) and subsequently characterized at its molecular level. The complete coding region of RfCRP is 672 bp in length, and encodes a protein containing 224 amino acids with a predicted molecular mass of 25.19 kD. Analysis of its derived amino acid sequence enabled typical features of pentraxin family members to be identified, including the pentraxin family signature in RfCRP. Results from multiple sequence alignment suggest the conservation of functionally important residues in RfCRP. According to the phylogenetic reconstruction that was generated using different pentraxin counterparts from different taxa, RfCRP shares a common vertebrate ancestral origin and most closely clusters with marine teleostan CRP. Furthermore, recombinant RfCRP demonstrated Ca2+-dependent agglutination activity against Escherichia coli, which could be completely inhibited in the presence of carbohydrates based ligands. Moreover, recombinant RfCRP also exhibited anti-bacterial activity against both E. coli and Streptococcus iniae. In addition, qPCR analysis indicated that RfCRP is ubiquitously expressed in physiologically important tissues, with pronounced expression in the spleen. After healthy fish were treated with polysaccharides or live S. iniae, basal expression of RfCRP was significantly upregulated in spleen and head kidney tissues. Collectively, our results suggest that RfCRP may be important in host anti-bacterial defense, and it might potentially participate in the acute phase of an infection.
Our second study on APPs was centered on gaining substantial insights into a SAA counterpart identified from rockfish, .especially regarding its expressional behavior under septic conditions. Serum amyloid A (SAA) is a vital protein involved in acute phase of an inflammation or infection. SAAs are released from hepatocytes under inflammatory conditions to protect healthy cells from being damaged by pathogens or from self-destructive mechanisms. In this study our previously constructed black rockfish (Sebastes schlegeli) cDNA library was used to identify the full-length cDNA sequence SAA homolog (RfSAA) and characterize at the molecular level. As expected, in-silico analysis of this homolog showed the typical domain architectures of their known counterparts. Open reading frame of RfSAA consisted of 313-bp DNA sequence. The derived polypeptide sequence of RfSAA had a 121-amino acid sequence with a molecular weight of 13 kD. Phylogenetic analysis as well as pairwise sequence alignment results showed that RfSAA was closely related to the Epinephelus coioides ortholog. Although RfSAA was expressed ubiquitously in the tissues analyzed, they were eminently expressed in liver tissue, suggesting their origin in hepatocytes. Quantitative real-time PCR analysis indicated that RfSAA can be significantly up-regulated by both bacterial and viral stimulation in liver tissues, affirming its putative importance in the acute phase of first-line host immune defenses.
In our third study, we identified and molecularly characterize a Hp counterpart from black rockfish, focusing on its expressional modulation under pathogenic stress. Hp is a positive acute phase protein in most of the animals, synthesized in liver, and its concentration is elevated during the acute phase of an infection. Hp binds with hemoglobin (Hb) during the hemolysis process to prevent the negative effects exerted by free Hb, including oxidative damage. Also here in, our previously constructed black rockfish (Sebastes schlegeli) cDNA library was used to identify the full-length cDNA sequences of Hp homolog (RfHp) and it was molecularly characterized. As expected, in silico analysis showed that RfHp houses the typical domain architecture of its known counterparts. Open reading frames of RfHp consisted of 942 bp DNA sequence. The derived polypeptide sequence of RfHp was composed of 313 amino acids (aa) with a predicted molecular weight of 34 kD. Phylogenetic analysis as well as pairwise sequence alignment results showed that RfHp was more closely related to its counterpart of Oreochromis mossambicus from an evolutionary perspective. Although RfHp was expressed ubiquitously in the tissues analyzed, it was particularly expressed in liver tissue, suggesting their origin in hepatocytes. Quantitative real-time PCR analysis indicated that RfHp was significantly up-regulated by both bacterial and viral stimulation in liver tissue, affirming its putative importance in the acute phase of the first-line host immune defenses.
In the second phase of the studies (described in chapter 2) we attempted to identify and characterize four distinct antioxidants from rock bream, two of which (catalase and thioredoxin reductase 3 (TrxR3)) are known to directly partake in antioxidant defense and other two (ferritin H and M) can indirectly participate in antioxidant defense by depriving the catalysts (Fe2+) of oxidative reactions such as 'Fenton' type reactions.
In the first study under chapter 2 we identified and characterized a catalase counterpart from rock bream. Catalases are known to be 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 (3,303 bp) of RbCat is comprised of a 1,581 bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kD 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 aa 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.
Our second study in this chapter was devoted to molecularly characterize a TrxR3 counterpart from rock bream. The thioredoxin (Trx) system plays a significant role in cellular antioxidative defense by dismutating the surpluses of reactive oxygen species. Thus, the role of thioredoxin reductase (TrxR) cannot be ignored, owing to its participation in initiating the Trx enzyme cascade. Here, we report the identification and molecular characterization of a teleostean TrxR (RbTrxR-3) ortholog that showed high similarity with the TrxR-3 isoforms of other vertebrates. The complete RbTrxR-3 coding sequence comprised 1800 nucleotides, encoding a 600-amino acid protein with a predicted molecular mass of ~ 66 kD. RbTrxR-3 consisted of 16 exons separated by 15 introns and had a total length of 12658 bp. In silico analysis of the RbTrxR-3 protein sequence revealed that it possesses typical TrxR domain architecture. Moreover, using multiple sequence alignment and pairwise sequence alignment strategies we showed that RbTrxR-3 has high overall sequence similarity to other teleostean TrxR-3 proteins, including highly conserved active site residues. Phylogenetic reconstruction of RbTrxR-3 affirmed its close evolutionary relationship with fish TrxR-3 orthologs, as indicated by its clustering pattern. RbTrxR-3 transcriptional analysis, performed using quantitative real time polymerase chain reaction (qPCR), showed that RbTrxR-3 was ubiquitously distributed, with the highest level of mRNA expression in the blood, followed by the gill, and liver. Live bacterial and viral stimuli triggered the modulation of RbTrxR-3 basal transcription in liver tissues that correlated temporally with that of its putative substrate, rock bream thioredoxin1 under same conditions of pathogenic stress. Finally, resembling the typical function of TrxR protein, purified recombinant RbTrxR-3 showed detectable dose-dependent thiol reductase activity against 5,5′-dithiobis (2-nitrobenzoic) acid. Taken together, these results suggest that RbTrxR-3 plays a role in the host Trx system under conditions of oxidative and pathogenic stress.
As the third study, we could identify the rock bream FerH like subunit, as crucial component of make up the ferritin complex. Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531 bp in length, encoding a 177 amino acid protein with a predicted molecular mass of 20.8 kD. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5′ untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195 bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense.
In final study under chapter 2, we described the characterization of FerM like subunit from rock bream. ferritin "M" subunit also contributes to form the ferritin nano-cage (24 mer) which possesses characteristic features of both the H and L subunits and was recently identified from 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 a 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 kD. 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 iridovirus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron binding activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H2O2-mediated DNA damage.
Collectively, this complete set of studies discovers selected putative APP counterparts and antioxidants exist in black rockfish and rock bream, respectively, further deciphering their expressional modulation at transcriptional level in response to pathogen stress. Moreover, some of the molecules were demonstrated to exert their expected functions in-vitro, suggesting their functional roles in fish physiology or immunity. Hence, the findings of our collective studies may help to betterment the progressive strategies which can enhance the survival of these fish against pathogen infection or oxidative stress.
Author(s)
Don Anushka Sandaruwan Elvitigala
Issued Date
2015
Awarded Date
2016. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007418
Alternative Author(s)
엘비티갈라, 돈 아누시카
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
Introduction 1
Chapter I. Identification and molecular characterization of three putative acute phase proteins from Black rock fish (Sebastes schlegelii) deciphering their roles in host acute phase response.
Part A - Characterization of a C-reactive protein (CRP) homologue from Black rockfish
1. Abstract 27
2. Materials and Methods 28
2.1. cDNA database of black rockfish 28
2.2 RfCRP sequence identification and profiling 29
2.3. Preparation of the RfCRP recombinant plasmid construct 29
2.4. Overexpression and purification of recombinant RfCRP (rRfCRP) 31
2.5. Determination of bacterial agglutination activity and its inhibition 31
2.6. Antibacterial activity analysis
2.6.1. Effect on growth of E. coli by overexpressing rRfCRP 32
2.6.2. Bacterial growth inhibition by purified rRfCRP 33
2.7. Fish husbandry and tissue collection 33
2.8. Immune stimulation 34
2.9. Total RNA isolation and cDNA synthesis 34
2. 10. Relative quantification of RfCRP mRNA expression 35
3. Results and Discussion 37
3.1. Delineation of sequence features and homology 37
3.2. Phylogenetic reconstruction 41
3.3. Integrity of purified rRfCRP 43
3.4. Bacterial agglutination activity 44
3.5. Ligand binding ability 45
3.6. Bacterial growth inhibition 47
3.7. Tissue specific transcription of RfCRP 50
3.8. Transcriptional modulation of RfCRP upon immune stimulation 51
4. Conclusion 54
Part B - Characterization of serum amyloid A (SAA) counterpart from Black rockfish
1. Abstract 56
2. Materials and Methods 57
2.1. Identification, sequence analysis and comparison 57
2.2. Experimental fish, husbandry and tissue collection 57
2.3. Immune challenge experiments 58
2.4. RNA isolation and first-strand cDNA synthesis 58
2.5. Transcriptional analysis by Quantitative real-time PCR (qPCR) and 58
statistical analysis
3. Results and Discussion 60
3.1. Sequence characteristics and homology 60
3.2. Phylogenetic position of RfSAA 62
3.3. Protein folding prediction of RfSAA 63
3.4. Tissue-specific expression of RfSAA 65
3.5. Expressional modulation in response to challenge experiments 66
4. Conclusion 69
Part C - Characterization of haptoglobin (Hp) counterpart from Black rockfish
1. Abstract 70
2. Materials and Methods 71
2.1. Identification, sequence analysis and comparison of RfHp and 71
RfSAA from the black rockfish
2.2. Experimental fish, husbandry and tissue collection 71
2.3. Immune challenge experiments 71
2.4. RNA isolation and first-strand cDNA synthesis 72
2.5. Transcriptional analysis by Quantitative real-time PCR (qPCR) and 72
statistical analysis
3. Results and Discussion 74
3.1. Sequence characterization of RfHp 74
3.2. Phylogenetic position of RfHp 77
3.3. Protein folding prediction of RfHp 77
3.4. Quantitative analysis of tissue-specific expression of RfHp 79
3.5. Quantitative analysis of expressional modulation in response to 80
challenge experiments
4. Conclusion 83
Chapter 2. Identification and molecular characterization of selected role players in host antioxidative defense from Rock bream (Oplegnathus fasciatus) portraying their functional significances. 84
Part A - Characterization of a catalase homologue from Rock bream
1. Abstract 84
2. Materials and Methods 86
2.1. Construction of rock bream cDNA sequence database 86
2.2. Identification of the complete cDNA sequence of RbCat 86
2.3. Identification of the complete genomic sequence of RbCat 86
2.4. In-silico characterization of RbCat 87
2.5. Expression and purification of recombinant RbCat (rRbCat) 88
2.6. Analysis of antioxidant activity of rRbCat 90
2.7. Evaluation of the biochemical properties of rRbCat 90
2.8. Experimental fish and tissue collection 91
2.9. Immune challenge experiments 92
2.10. Total RNA extraction and cDNA synthesis 92
2.11. RbCat transcriptional analysis by reverse transcription PCR 93
followed by quantitative real time PCR
3. Results and Discussion 94
3.1. Sequence characterization and phylogenetic relationship of RbCat 94
3.2. Modeled tertiary structure of RbCat 99
3.3. Genomic architecture and predicted core promoter region of RbCat 100
3.4. Protein expression and purification of rRbCat 104
3.5. Antioxidant activity of rRbCat 105
3.6. Biochemical properties of rRbCat 106
3.7. Tissue-specific mRNA expression profile of RbCat 109
3.8. Transcriptional response of RbCat upon immune stimulation 111
4. Conclusion 114
Part B - Characterization of a thioredoxin reductase 3 (TrxR3) homologue from
Rock bream
1. Abstract 116
2. Materials and Methods 117
2.1. Identification of complete RbTrxR-3 cDNA and genomic DNA 117
Sequences
2.2. RbTrxR-3 sequence profiles and phylogenetic analyses 118
2.3. Molecular cloning, over-expression, and purification of 119
recombinant RbTrxR-3 (rRbTrxR-3)
2.4. rRbTrxR-3 thiol reductase activity analysis 120
2.5. Animal husbandry and tissue collection 120
2.6. Pathogen challenge experiments 121
2.7. Total RNA extraction and cDNA synthesis 122
2.8. Measurement of RbTrxR-3 and rock bream Trx-1 (RbTrx-1) 122
mRNA expression levels in liver tissues by using quantitative PCR
3. Results and Discussion 123
3.1. Molecular properties and sequence similarity of RbTrxR-3 123
3.2. Comparative analysis of RbTrxR-3 gene architecture 128
3.3. Phylogenetic reconstruction of RbTrxR-3 129
3.4. Integrity of purified rRbTrxR-3 131
3.5. Thiol-reductase activity of RbTrxR-3 132
3.6. Distribution of RbTrxR-3 mRNA in rock bream tissues 133
3.7. Temporal transcriptional response of RbTrxR-3 to live pathogenic 135
Stimuli
4. Conclusion 137
Part C - Characterization of ferritin H (FerH) subunit from rock bream
1. Abstract 139
2. Materials and Methods 140
2.1. Identification and sequence analysis of RbFerH 140
2.2. Expression and purification of recombinant RbFerH fusion protein 141
2.3. Iron(II) depriving activity of rRbFerH 143
2.4. Antibacterial activity of rRbFerH 144
2.5. Determination of the effect of rRbFerH on oxidative damage of DNA 144
2.6. Experimental fish and tissue collection 145
2.7. Immune challenge experiments 146
2.8 Total RNA extraction and cDNA synthesis 146
2.9 RbFerH transcriptional analysis by quantitative real-time PCR 147
3. Results and Discussion 148
3.1. Sequence profiles and homology 148
3.2. Phylogenetic relationship of RbFerH 154
3.3. Integrity, purity and degree of ferritin complex formation of rRbFerH 156
3.4. Iron(II) depriving activity of rRbFerH 157
3.5. Antibacterial activity of rRbFerH 159
3.6. Protective effect of rRbFerH in oxidative DNA damage 160
3.7. Spatial expression pattern of RbFerH 162
3.8. Transcriptional modulation of RbFerH expression upon immune 163
stimulation
4. Conclusion 167
Part D - Characterization of ferritin M (FerM) subunit from rock bream
1. Abstract 168
2. Materials and Methods 169
2.1 Identification and sequence profiling 169
2.2 Overexpression and purification of the recombinant RbFerM fusion 169
Protein
2.3. Effect of rRbFerM protein concentration on the iron binding activity 172
2.4. Effect of temperature on the iron binding activity of rRbFerM 172
2.5. Determination of the DNA protection effect of rRbFerM under 173
oxidative stress
2.6. Experimental fish and tissue collection 173
2.7. Immune challenge experiments 174
2.8 Total RNA extraction and cDNA synthesis 175
2.9 RbFerM transcriptional analysis by quantitative real time PCR 175
3. Results and Discussion 177
3.1. Sequence characterization of RbFerM 177
3.2. Evolutionary proximity of RbFerM with its orthologs 181
3.3. Integrity and purity of overexpressed recombinant RbFerM 182
(rRbFerM)
3.4. Iron binding activity for different concentrations of rRbFerM 184
3.5. Iron binding activity of rRbFerM as a function of temperature 185
3.6. DNA protection effect of rRbFerM under oxidative stress 186
3.7. Transcriptional distribution of RbFerM in selected tissues 188
3.8. Transcriptional behavior of RbFerM upon immune stimulation 190
4. Conclusion 195
General Conclusion 196
References 198
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Don Anushka Sandaruwan Elvitigala. (2015). Transcriptome wide discovery of selected role players in acute phase response of black rockfish (Sebastes schlegelii) and antioxidative defense of rock bream (Oplegnathus fasciatus), revealing their putative significance in host immune responses
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