유전자 재조합 단백질 Adenylate Kinase, Nucleoside Diphosphate Kinase와 Heat-Shock Protein 70의 결핵균에 대한 방어면역효능 분석

이승헌,이은계,김수연,조상래,박영길,배길한,Lee, Seung-Heon,Lee, Eun-Gae,Kim, Su-Yeon,Cho, Sang-Nae,Park, Young-Kil,Bai, Gill-Han 대한결핵및호흡기학회 2005 Tuberculosis and Respiratory Diseases Vol.58 No.2

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant. 배 경 : 최근 결핵에 대한 새로운 백신 개발은 초회 면역 방법 및 추가 면역 방법을 이용하는 방향으로 연구되고 있다. 본 실험은 새로운 백신 후보 물질로서의 가능성을 알아보기 위하여 결핵균 adenylate kinase (AK), nucleoside diphosphate (NdK) 및 heat shock protein 70(Hsp70)의 결핵균에 대한 방어면역효능을 측정하였다. 방 법 : 재조합 단백질들을 정제하기 위하여 중합효소 연쇄반응으로 증폭한 결핵균 유전자 단편들을 E.coli expression vector, pQE30에 클로닝한 후, Ni-NTA resin을 이용하여 정제하였다. DDA와 재조합 단백질들을 마우스에 면역주사하고 면역반응 생성 유무를 확인하기 위하여 항체와 $IFN-{\gamma}$ 생성능을 측정하였다. 면역주사 한 마우스에 결핵균을 공기 감염시킨 후, 폐와 비장을 분리하여 결핵균 생균수 실험을 하였다. 결 과 : 재조합 단백질 AK, NdK 와 Hsp70을 면역보강제인 DDA를 이용하여 면역주사 한 결과에서, 생리식염수 혹은 DDA를 면역주사 한 마우스에 비교하여 재조합 단백질을 면역주사 한 마우스에서는 각 항원에 대해 항체와 $IFN-{\gamma}$ 생성능이 높게 나타났으나 결핵균에 대한 효과적인 방어면역효능은 나타나지 않았다. 결 론 : 마우스를 모델로 한 결핵균에 대한 방어면역효능 실험에서, 면역보강제 DDA를 이용한 재조합 단백질 AK, NdK 및 Hsp70을 면역주사 한 경우에는 결핵균의 성장을 효과적으로 조절하지 못하였다. 혼합 단백질 혹은 다른 T세포 면역보강제의 사용에 의한 추시가 필요하다.

유전자 재조합 단백질 Adenylate Kinase, Nucleoside Diphosphate Kinase와 Heat-Shock Protein 70의 결핵균에 대한 방어면역효능 분석

이승헌 ( Seung Heon Lee ),이은계 ( Eun Gae Lee ),김수연 ( Su Yeon Kim ),조상래 ( Sang Nae Cho ),박영길 ( Young Kil Park ),배길한 ( Gill Han Bai ) 대한결핵 및 호흡기학회 2005 Tuberculosis and Respiratory Diseases Vol.58 No.2

배경 : 최근 결핵에 대한 새로운 백신 개발은 초회 면역 방법 및 추가 면역 방법을 이용하는 방향으로 연구되고 있다. 본 실험은 새로운 백신 후보 물질로서의 가능성을 알아보기 위하여 결핵균 adenylate kinase (AK), nucleoside diphosphate (NdK) 및 heat shock protein 70 (Hsp70)의 결핵균에 대한 방어면역효능을 측정하였다. 방법 : 재조합 단백질들을 정제하기 위하여 중합효소 연쇄반응으로 증폭한 결 Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kina

NDP kinase 2 regulates expression of antioxidant genes in Arabidopsis

Yang, Kyung-Ae,Moon, Hae-Jeong,Kim, Gyu-Tae,Lim, Chan-Ju,Hong, Jong-Chan,Lim, Chae-Oh,Yun, Dae-Jin Plant molecular biology and biotechnology research 2003 Plant molecular biology and biotechnology research Vol.2003 No.-

NDP kinases (NDPKs) are multifunctional proteins involved in cell proliferation, development, and differentiation in eukaryotes. Previously, we reported that Arabidopsis NDP kinase2 (AtNDPK2) is a component of H_(2)O_(2)-activated MAPK signaling in plants and demonstrated that its over-expression in plants alters cellular redox conditions (Proc.Natl.Acad.Sci.U.S.A.,100,358-363,2003). To elucidate how AtNDPK2 can regulate cellular redox state, we analyzed gene expression profiles of transgenic plants over-expressing AtNDPK2 using cDNA microarray technology. Constitutive over-expression of AtNDPK2 in plants induced numerous genes including those involved in cellular signal transduction and protection, suggesting that NDPKs are also multifunctional in plants. Among the induced genes, we further analyzed the expression of antioxidant genes by Northern blot analysis. Expression of peroxidase, catalase, thioredoxin, thioredoxin reductase, and peroxiredoxin was significantly increased in transgenic plants over-expressing AtNDPK2. Furthermore, the gene expression pattern obtained by Northern blotting was quantitatively consistent with the cDNA microarray analysis. Based on these observations, we suggest that down regulation of cellular redox state in AtNDPK2 transgenic plants is mediated by genes involved in antioxidant and protective processes.

Bradykinin Receptor의 발현에 미치는 녹농균유래 Nucleoside Diphosphate Kinase 및 Flagellin의 효과

김용재,신희성,하운환,Kim, Yong-Jae,Shin, Hee-Sung,Jin, Shouguang,Ha, Un-Hwan 한국미생물학회 2014 미생물학회지 Vol.50 No.4

Immune defense responses against Pseudomonas aeruginosa infection play an important role in maintaining homeostasis in the human body. Previously, we reported that expression of the bradykinin receptor (BR) is induced in response to P. aeruginosa infection. However, the factors responsible for the induction was uncertain. Here, we found that the type III secretion system (T3SS) is responsible for the induction of BR expression, and nucleoside diphosphate kinase (Ndk), as a novel T3SS effector, mediates the upregulation. The Ndk-mediated expression of BR was not induced by fliC mutant treatment, indicating the involvement of flagellin, one of the well-known pathogen-associated molecular patterns (PAMPs). Taken together, this study demonstrated that Ndk cooperates with flagella in the development of defense responses against P. aeruginosa infection. 병원성 균주인 녹농균(Pseudomonas aeruginosa) 감염에 대응하여 나타나는 면역반응은 인체의 항상성 유지에 중요하다. 선행 연구에서 녹농균의 감염에 대응하여 bradykinin receptor (BR)의 발현이 증가됨을 보고하였지만, 발현유도에 관여하는 녹농균 유래인자에 대해서는 보고한 바가 없었다. 이번 연구에서는 녹농균에 의한 BR의 발현은 Type III secretion system (T3SS)이 관여하지만, 기존에 알려진 T3SS인자가 아닌 nucleoside diphosphate kinase (Ndk)에 의한 것으로 조사되었다. 하지만 pDNNDK를 이용한 transfection 실험 결과, Ndk 만으로는 BR의 발현이 유도되지 않았으며, Ndk와 함께 flagella가 필요함을 발견하였다. 이러한 결과는 기존에 보고된 주요 pathogen-associated molecular patterns (PAMPs)인 flagella와 더불어 감염대응에 관여하는 Ndk를 발굴한 의미가 있으며, 녹농균에 의한 질병기전을 이해하는데 도움을 줄 수 있다.

Transgenic poplar expressing AtNDPK2 exhibits enhanced biomass in the LMO field

Chul-Han An,김윤희,Sung-Chul Park,Jae Cheol Jeong,Haeng-Soon Lee,Yong Im Choi,Eun Woon Noh,윤대진,Se-Bin Kim,Sang-Soo Kwak 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.3

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes and auxin-responsive genes in plants. Previously, it was noted that the overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2promoter in transgenic poplar (Populus alba × P. tremular var. glandulosa) plants (referred to as SN plants) enhanced tolerance to oxidative stress and improved growth (Plant Biotechnol J 9: 34-347, 2011). In this study, growth of transgenic poplar was assessed under living modified organism (LMO) field conditions in terms of biomass in the next year. The growth of transgenic poplar plants increased in comparison with non-transgenic plants. The SN3 and SN4 transgenic lines had 1.6 and 1.2 times higher dry weight in stems than non-transgenic plants at 6 months after planting, respectively. Transgenic poplar also exhibited increased transcript levels of auxin-response genes such as IAA1, IAA2, IAA5 and IAA6. These results suggest that enhanced AtNDPK2 expression increases plant biomass in transgenic poplar through the regulation of auxin-response genes.

Transgenic poplar expressing <i>Arabidopsis NDPK2</i> enhances growth as well as oxidative stress tolerance

Kim, Yun‐,Hee,Kim, Myoung Duck,Choi, Young Im,Park, Sung‐,Chul,Yun, Dae‐,Jin,Noh, Eun Woon,Lee, Haeng‐,Soon,Kwak, Sang‐,Soo Blackwell Publishing Ltd 2011 Plant biotechnology journal Vol.9 No.3

<P><B>Summary</B></P><P>Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of <I>Arabidopsis NDPK2</I> (<I>AtNDPK2</I>) under the control of an oxidative stress‐inducible <I>SWPA2</I> promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (<I>Populus alba </I>× <I>Poplus glandulosa</I>) expressing the <I>AtNDPK2</I> gene under the control of a <I>SWPA2</I> promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of <I>AtNDPK2</I> expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant’s tolerance to MV‐mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV‐treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin‐response genes <I>IAA2</I> and <I>IAA5</I>. These results suggest that enhanced <I>AtNDPK2</I> expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.</P>

Transgenic poplar expressing AtNDPK2 exhibits enhanced biomass in the LMO field

An, Chul-Han,Kim, Yun-Hee,Park, Sung-Chul,Jeong, Jae-Cheol,Lee, Haeng-Soon,Choi, Yong-Im,Noh, Eun-Woon,Yun, Dae-Jin,Kim, Se-Bin,Kwak, Sang-Soo The Korean Society of Plant Biotechnology 2011 식물생명공학회지 Vol.38 No.3

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes and auxin-responsive genes in plants. Previously, it was noted that the overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic poplar (Populus alba ${\times}$ P. tremular var. glandulosa) plants (referred to as SN plants) enhanced tolerance to oxidative stress and improved growth (Plant Biotechnol J 9: 34-347, 2011). In this study, growth of transgenic poplar was assessed under living modified organism (LMO) field conditions in terms of biomass in the next year. The growth of transgenic poplar plants increased in comparison with non-transgenic plants. The SN3 and SN4 transgenic lines had 1.6 and 1.2 times higher dry weight in stems than non-transgenic plants at 6 months after planting, respectively. Transgenic poplar also exhibited increased transcript levels of auxin-response genes such as IAA1, IAA2, IAA5 and IAA6. These results suggest that enhanced AtNDPK2 expression increases plant biomass in transgenic poplar through the regulation of auxin-response genes.

Nucleoside Diphosphate Kinase from Microorganisms

Chul Hee Choi 대한미생물학회 2013 Journal of Bacteriology and Virology Vol.43 No.2

Oligomeric Structures Determine the Biochemical Characteristics of Human Nucleoside Diphosphate Kinases

Kim, Sun-Young,Song, Eun-Joo,Chang, Keun-Hye,Kim, Eun-Hee,Chae, Suhn-Kee,Lee, Han-Soo,Lee, Kong-Joo Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.4

Major human Nucleoside diphosphate kinases (NDPKs) exist as hetero-oligomers, consisting of NDPK-A and NDPK-B, rather than homo-oligomer. To investigate their biological function depending on the oligomeric structure in vivo, we characterized the biochemical properties of cellular NDPK. Cellular NDPKs, which are made up of a unique combination of isoforms, were purified from human erythrocyte and placenta. We found that cellular NDPK and recombinant isoforms NDPKs have their own distinct biochemical properties in autophosphorylation, stability toward heat or urea, and DNA binding. Cellular NDPK was found to have unique characteristics rather than the expected additive properties of recombinant isoforms. The mutations in the dimeric interface of NDPK-B (R34G, N69H or K135L) caused defective DNA binding and simultaneously reduced the enzymatic stability These results suggest that the oligomeric interaction could play a major role in the stability of catalytic domain and might be related to the regulation of various cellular functions of NDPK.

Quaternary structural analysis of nucleoside diphosphate kinases using capillary electrophoresis

Heo, Yoo Jeong,Kim, Sun Young,Kim, Eunhee,Lee, Kong-Joo 梨花女子大學校 藥學硏究所 1998 藥學硏究論文集 Vol.- No.7

Capillary electrophoresis was used to monitor the quaternary structure and structural changes by denaturants of nucleoside diphosphate kinase (NDP kinase). NDP kinase from human erythrocyte consists of two kinds of polypeptide chains : A (Nm23-H1)and B(Nm23-H2), which are the products of nm23-H1 and -H2 genes, respectively. Structural characteristics of native NDP kinase, recombinant Nm23-H1 and -H2 were examined using capillary electrophoresis employing high pH running buffer in an uncoated fused-silica capillary as a separation column. The results were compared with those of the conventional sodium dodecyl sulfate and non-denaturing polyacrylamide gel electrophoresis. It shows that capillary electrophoresis is a possible tool to investigate protein structure, including the quaternary structure. This protocol was applied to the investigation of the denaturation process of each enzyme by denaturants (6 M urea or heating th 70℃) and to the monitoring of the interaction between denatured Nm23-H1 and -H2. The structural information of NDP kinase obtained by analysis using capillary electrophoresis is valuable for understanding its suggested biological function as a tumor metastasis suppressor and c-myc transcription factor, etc.