酵母表达的长角血蜱谷氨酰胺转移酶及其活性分析

doi:10.11843/j.issn.0366-6964.2017.02.017

Abstract

Abstract:

Transglutaminases (TGases) are a widely distributed group of enzymes that catalyze the formation of isopeptide bonds either through protein cross-linking via ε-(γ-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide- bound glutamine residues, and involved in multiple important physiological events. This study was aimed at cloning and expression of a transglutaminase gene from a tick Haemaphysalis longicornis Shanghai strain (HlTGase, GenBank accession number: KX59300), and analysis of the molecular characterization and the enzyme activity of the recombinant HlTGase. Total RNA of the adult ticks was extracted and HlTGase gene was amplified. The open reading frame of HlTGase was inserted into a plasmid pPICZC and transformed electrically into yeast Pichia pastoris, which was then induced to express the recombinant HlTGase. The result showed that the open reading frame of the gene was 2 262 bp, which encoded a polypeptide of 756 aa. The polypeptide possessed four transglutaminase domains. The calculated molecular weight of the polypeptide was 84.6 kDa. A phylogenetic tree showed that the polypeptide had a closest relationship with that of Drosophila melanogaster among TGases from 11 typical species, which was correspondent with the traditional taxonomical status. The antibody against the recombinant HlTGase recognized the endogenous HlTGase in a Western blotting analysis. The results also showed that the recombinant HlTGase had enzyme activity to catalyze cross linking between proteins. However, the activity was lower than a commercial available TGase from guinea pigs. HlTGase was expressed in yeast successfully. More modification in the expression and purification of the recombinant HlTGase might be required to improve the enzyme activity. This study would provide basic information for further study on the function and potential application of HlTGase.

CLC Number:

Q78
S852.746

HU Yan-hong, YU Ling-ying, HUANG Xiao-hong. Analysis on a Transglutaminase from Haemaphysalis longicornis Expressed in Pichia pastoris[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 331-339.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2017.02.017

https://www.xmsyxb.com/EN/Y2017/V48/I2/331

References

［1］孟真，姜理平，陆群英，等. 一组长角血蜱中检出莱姆病螺旋体和斑点热群立克次体复合感染［J］. 中华流行病学杂志，2008，29(12)：1217-1220.
MENG Z, JIANG L P, LU Q Y, et al. Detection of co-infection with Lyme spirochetes and Spotted fever group rickettsiae in a group of Haemaphysalis longicornis ［J］. Chinese Journal of Epidemiology, 2008, 29(12):1217-1220. (in Chinese)
［2］SUN J, LIN J, GONG Z, et al. Detection of spotted fever group Rickettsiae in ticks from Zhejiang province, China ［J］. Exp Appl Acarol, 2015, 65(3): 403-411.
［3］SHEN Y, GAO J, XU K, et al. Babesiasis in Nanjing area, China ［J］. Trop Anim Health Prod, 1997, 29(4 Suppl): 19S-22S.
［4］IKADAI H, SASAKI M, ISHIDA H, et al. Molecular evidence of Babesia equi transmission in Haemaphysalis longicornis ［J］. Am J Trop Med Hyg, 2007, 76(4): 694-697.
［5］FOLK J E, FINLAYSON J S. The epsilon-(gamma-glutamyl) lysine crosslink and the catalytic role of transglutaminases［J］. Adv Protein Chem, 1977, 31: 1-133.
［6］LORAND L, GRAHAM R M. Transglutaminases: crosslinking enzymes with pleiotropic functions［J］. Nat Rev Mol Cell Biol, 2003, 4(2): 140-156.
［7］ELLI L, BRGAMINI C M, BARDELLA M T, et al. Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver ［J］. Dig Liver Dis, 2009, 41(8): 541-550.
［8］COLLIGHAN R J, GRIFFIN M. Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications ［J］. Amino Acids, 2009, 36(4): 659-670.
［9］BASSO M, BERLIN J, XIA L, et al. Transglutaminase inhibition protectes against oxidative stress-induced neuronal death downstream of pathological ERK activation ［J］. J Neurosci, 2012, 32(19):6561-6569.
［10］CHANDRASHEKAR R, MEHTA K. Transglutaminase-catalyzed reactions in the growth, maturation and development of parasitic nematodes ［J］. Parasitol Today, 2000, 16(1): 11-17.
［11］MILANI A, VECCHIETTI D, RUSMINI R, et al. TgpA, a protein with a eukaryotic-like transglutaminase domain, plays a critical role in the viability of Pseudomonas aeruginosa［J］. PLoS One, 2012, 7(11): e50323.
［12］HALL M, WANG R, VAN ANTWERPEN R, et al. The crayfish plasma clotting protein: a vitellogenin-related protein responsible for clot formation in crustacean blood ［J］. Proc Natl Acad Sci U S A, 1999, 96(5): 1965-1970.
［13］WANG R, LIANG Z, HAL M, et al. A transglutaminase involved in the coagulation system of the freshwater crayfish, Pacifastacus leniusculu. Tissue localisation and cDNA cloning［J］.Fish Shellfish Immund, 2001,11(7):623-637.
［14］CHAIKEERATISAK V, SOMBOONWIWAT K, TASSANAKAJON A. Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots［J］. PLoS One, 2012, 7(10): e47384.
［15］FAGUTAO F F, MANINGAS M B, KONDO H, et al. Transglutaminase regulates immune-related genes in shrimp［J］. Fish Shellfish Immunol, 2012, 32(5): 711-715.
［16］OSAKI T, OKINO N, TOKUNAGA F, et al. Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin ［J］. J Biol Chem, 2002, 277(42): 40084-40090.
［17］MATSUDA Y, KOSHIBA T, OSAKI T, et al. An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh ［J］. J Biol Chem, 2007, 282(52): 37316-37324.
［18］MEANINGS M B, KONDO H, HIRONO I, et al. Essential function of transglaminase and clotting protein in shrimp immunity［J］. Mol Immunol, 2008, 45(5): 1269-1275.
［19］MATSUDA Y, OSAKI T, HASHII T, et al. A cysteine-rich protein from an arthropod stabilizes clotting mesh and immobilizes bacteria at injury sites ［J］. J Biol Chem, 2007, 282(46): 33545-33552.
［20］THEOPOLD U, SCHMIDT O, SÖDERHÄLL K, et al. Coagulation in arthropods: defence, wound closure and healing ［J］. Trends Immunol, 2004, 25(6): 289-294.
［21］SINGER M A, HORTSCH M, GOODMAN C S, et al. Annulin, a protein expressed at limb segment boundaries in the grasshopper embryo, is homologous to protein cross-linking transglutaminases ［J］. Develop Biol, 1992, 154(1): 143-159.
［22］LIN X, SÖDERHÄLL K, SÖDERHÄLL L. Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis ［J］. BMC Immunol, 2008, 9:58.
［23］周金林，周勇志，龚海燕，等. 我国长角血蜱孤雌生殖种群的发现和生物学特性的研究［J］. 中国媒介生物学及控制杂志，2004，15（3）：173-174.
ZHOU J L, ZHOU Y Z, GONG H Y, et al. Discovery of parthenogenesis population of Haemaphysalis longicornis in China and its biological characteristic ［J］. Chinese Journal of Vector Biology and Control, 2004，15(3)：173-174. (in Chinese)
［24］HUANG X, TSUJI N, MIYOSHI T, et al. Characterization of glutamine:fructose-6-phosphate aminotransferase from the ixodid tick, Haemaphysalis longicornis, and its critical role in host blood feeding ［J］. Int J Parasitol, 2007, 37(3-4): 383-392.
［25］LIN Q, YANG M, HUANG Z, et al. Cloning, expression and molecular characterization of a 14-3-3 gene from a parasitic ciliate, Cryptocaryon irritans［J］. Vet Parasitol, 2013, 197(3-4): 427-435.
［26］SLAUGHTER T F, ACHYUTHAN K E, LAI T S, et al. A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate［J］.Anal Biochem, 1992, 205(1): 166-171.
［27］TRAORE F, MEUNIER J C. Cross-linking of caseins by human placental factor XIII［J］. J Agric Food Chem, 1991, 39(10): 1892-1896.
［28］ELLI L, CIULLA M M, BUSCA G, et al. Beneficial effects of treatment with transglutaminase inhibitor cystamine on the severity of inflammation in a rat model of inflammatory bowel disease［J］. Lab Invest, 2011, 91(3):452-461.
［29］YEH M S, TSAI W L, CHENG W. Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation［J］.Fish Shellfish Immunol, 2013, 35(5):1613-1623.

[1]	DAI Xueyu, ZHANG Qianyi, XU Lu, ZHAO Qizu, WANG Qin, XIA Yingju. Research Progress and Application of CRISPR/Cas9 Gene Editing Technology in Prevention and Control of Important Swine Virus Diseases [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 943-951.
[2]	WU Jinyan, TIAN Hong, MENG Xuelian, HUI Xiaoting, WANG Yaojie, GUAN Yuhua, SHANG Youjun, LIU Yongsheng, ZHANG Zhidong. Establishment of Vero/SLAM Cell Lines with Stable Expression SLAM to Enhanced PPRV Replication Using the Lentiviral Expression System [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 218-226.
[3]	HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHANG Jing, ZHANG Wei-feng, XI Jian-zhong. Cloning and Transcriptional Activity of Swine StAR Gene Promoter [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1524-1532.
[4]	HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHAO Si-si, ZHANG Wei-feng, ZHANG Jun, ZHANG Jian-ting, BIAN Hui-min. Cloning and Activity Analysis of the Promoter Region of Swine DKK1 Gene [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1150-1157.
[5]	XU Qian-ru, ZHANG Er-qin, GUO Jun-qing, YANG Ji-fei, LIU Yun-chao, WANG Ai-ping, WANG Li, WAN Bo, WANG Lei, JIANG Da-wei, DENG Rui-guang, ZHANG Gai-ping. Expression and Detection of NDV-F Protein in Rice Endosperm [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1167-1172.
[6]	HE Zu-yong，LIU Zhi-guo，SUN Yan-xia，CONG Pei-qing，CHEN Yao-sheng. Generation of a Transgenic Mouse Model Co-expressing Three Fibrolytic Enzyme Genes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1162-1169.
[7]	HU Xiang-yun,GAO Xiao-long，FU Xiang-jing，LIU Dan-dan，FAN Wen-tao，WANG Yan-ping，DU En-qi，WANG Xing-long，DANG Ru-yi，YANG Zeng-qi. Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(6): 1071-1076.
[8]	FU Mei，CHEN Ya-bing，LI Jian，XIONG Xian-rong，LAN Dao-liang. NOBOX Gene Expression in Maiwa Yak Oocytes，Early Embryogenesis and Fetal Ovaries [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1746-1751.
[9]	LIU Zhi-guo. Research Progress on CRISPR/Cas9 Mediated Genome Editing [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(10): 1567-1583.
[10]	CHEN Li，ZHAO Wei，ZHAN Si-yuan，LI Dan，LI Li，ZHONG Tao，WANG Lin-jie，ZHANG Hong-ping. The Expression Stability Analysis of Reference Genes in the Different Tissues and Skeletal Muscle of Different Development Periods in Goat [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1228-1236.
[11]	ZHANG Chun-xiang，ZHAO Hui，ZHANG Guo-lin，GUO Li-na，REN You-she. The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1237-1245.
[12]	WANG Jing-jiao，ZHANG Ming-yue，TAN Bei-bei，ZHANG Cui，LI Dong-jie，DAI Yun-ping，LI Ning,LI Shi-jie. Integration Site Detection of Exogenous Gene in Transgenic Cloned Cattle [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 553-558.
[13]	JI Yuan-kai,TIAN Xue，FAN Rui-wen，YU Xiu-ju,ZHAO Yuan-yuan，MA Shu-hui，DONG Chang-sheng. Study on the Expression and Localization of MATP in Different Colors of Alpaca Skin and the Correlation Analysis of Other Genes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(3): 502-508.
[14]	WANG Xiang-peng, WEI Rui-fang, XIAO Shu-qi, ZHOU En-min. Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163 by Lentiviral Vector for the Production of Porcine Reproductive and RespiratorySyndrome Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1797-1804.
[15]	HE Sheng-fang, FU Xiang-jing, LIU Yang-kun, MU Guo-hui, LIU Hai-jin,WANG Xing-long, DU En-qi, YANG Zeng-qi. Establishing a Method for Highly Efficient Producing Nanometer Antibodies [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(9): 1487-1493.

Analysis on a Transglutaminase from Haemaphysalis longicornis Expressed in Pichia pastoris

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments