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http://dx.doi.org/10.13000/JFMSE.2016.28.4.903

Cloning, Expression Analysis and Enzymatic Characterization of Cathepsin L from the Inshore Hagfish (Eptatretus burgeri)  

JANG, Jin-Hyeon (Pukyong National University)
SON, So-Hee (Pukyong National University)
JO, Hyeon-Kyeong (Pukyong National University)
CHUNG, Joon-Ki (Pukyong National University)
LEE, Hyung-Ho (Pukyong National University)
Publication Information
Journal of Fisheries and Marine Sciences Education / v.28, no.4, 2016 , pp. 903-912 More about this Journal
Abstract
Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.
Keywords
Cathepsin L; Cysteine protease; Eptatretus burgeri;
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1 Barrett, A. J. & Kirschke, H.(1981). cathepsin B, cathepsin H, and cathepsin L. Methods in Enzymology, 80, 535-561.   DOI
2 Barrett A. J..Rawlings N. D..Woessner J. F. Jr.(Eds.)(1998) Hand book of Proteolytic Enzymes, Academic Press, London, 258.
3 Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.   DOI
4 Mallatt, J. & Sullivan, J.(1998). 28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes. Molecular Biology and Evolution, 15(12), 1706-1718.   DOI
5 McDonald, J. K.(1985). An overview of protease specificity and catalytic mechanisms: Aspects related to nomenclature and classification. The Histochemical Journal, 17(7), 773-785.   DOI
6 Oisi, Y..Ota, K. G..Kuraku, S..Fujimoto, S. & Kuratani, S.(2013). Craniofacial development of hagfishes and the evolution of vertebrates. Nature, 493(7431), 175-180.   DOI
7 Petersen, T. N..Brunak, S..von Heijne, G. & Nielsen, H.(2011). SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nature Methods, 8(10), 785-786.   DOI
8 Shahinian, H..Tholen, S. & Schilling, O.(2013). Proteomic identification of protease cleavage sites: Cell-biological and biomedical applications. Expert Review of Proteomics, 10(5), 421-433.   DOI
9 Song, G..Bailey, D. W..Dunlap, K. A..Burghardt, R. C..Spencer, T. E..Bazer, F. W. & Johnson, G. A.(2010). Cathepsin B, cathepsin L, and cystatin C in the porcine uterus and placenta: Potential roles in endometrial/placental remodeling and in fluid-phase transport of proteins secreted by uterine epithelia across placental areolae. Biology of Reproduction, 82(5), 854-864. doi:10.1095/biolreprod.109.080929 [doi]   DOI
10 Tingaud-Sequeira, A. & Cerda, J.(2007). Phylogenetic relationships and gene expression pattern of three different cathepsin L (ctsl) isoforms in zebrafish: Ctsla is the putative yolk processing enzyme. Gene, 386(1), 98-106.   DOI
11 Guo, Y. L..Kurz, U..Schultz, J..Lim, C. C..Wiederanders, B. & Schilling, K.(2000). The ${\alpha}1/2$ helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily. FEBS Letters, 469(2), 203-207.   DOI
12 Dacks, J. B..Kuru, T..Liapounova, N. A. & Gedamu, L.(2008). Phylogenetic and primary sequence characterization of cathepsin B cysteine proteases from the oxymonad flagellate monocercomonoides. Journal of Eukaryotic Microbiology, 55(1), 9-17.   DOI
13 Duncan, E. M..Muratore-Schroeder, T. L..Cook, R. G..Garcia, B. A..Shabanowitz, J..Hunt, D. F. & Allis, C. D.(2008). Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation. Cell, 135(2), 284-294.   DOI
14 Fonovic, M. & Turk, B.(2014). Cysteine cathepsins and their potential in clinical therapy and biomarker discovery. PROTEOMICS-Clinical Applications, 8(5-6), 416-426.   DOI
15 Hall, T.(2004). BioEdit version 7.0. 0. Distributed by the Author, Website: www.Mbio.Ncsu.Edu/BioEdit/Bioedit.Html,
16 Kisselev, A. F..Akopian, T. N..Castillo, V. & Goldberg, A. L.(1999). Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown. Molecular Cell, 4(3), 395-402.   DOI
17 Kumar, S..Nei, M..Dudley, J. & Tamura, K.(2008). MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics, 9(4), 299-306. doi:10.1093/bib/bbn017 [doi]   DOI
18 Kuraku, S..Hoshiyama, D..Katoh, K..Suga, H. & Miyata, T.(1999). Monophyly of lampreys and hagfishes supported by nuclear DNA Coded genes. Journal of Molecular Evolution, 49(6), 729-735.   DOI
19 Turk, V..Stoka, V..Vasiljeva, O..Renko, M..Sun, T..Turk, B. & Turk, D.(2012). Cysteine cathepsins: From structure, function and regulation to new frontiers. Biochimica Biophysica Acta (BBA)-Proteins and Proteomics, 1824(1), 68-88.   DOI
20 Turk, B..Turk, D. & Turk, V.(2000). Lysosomal cysteine proteases: More than scavengers. Biochimica Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1477(1), 98-111.   DOI
21 Vernet, T..Berti, P. J..de Montigny, C..Musil, R..Tessier, D. C..Menard, R..Thomas, D. Y.(1995). Processing of the papain precursor The ionization state of a conserved amino-acid motif within the pro region participates in the regulation of intramolecular processing. Journal of Biological Chemistry, 270(18), 10838-10846.   DOI
22 Wang, Z..Dove, P..Wang, X..Shamas-Din, A..Li, Z..Nachman, A..Climie, S.(2015). FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing. Cell Death & Disease, 6(7), e1815.   DOI
23 Xu, Z. J..Nakajima, M..Suzuki, Y. & Yamaguchi, I.(2002). Cloning and characterization of the abscisic acid-specific glucosyltransferase gene from adzuki bean seedlings. Plant Physiology, 129(3), 1285-1295. doi:10.1104/pp.001784 [doi]   DOI
24 Zavasnik Bergant, T. & Turk, B.(2006). Cysteine cathepsins in the immune response. Tissue Antigens, 67(5), 349-355.   DOI
25 Zhou, J..Zhang, Y..Li, Q. & Cai, Z.(2015). Evolutionary history of cathepsin L (L-like) family genes in vertebrates. International Journal of Biological Sciences, 11(9), 1016.   DOI