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Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development

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Abstract

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1–25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.

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References

  • Alvarez-González CA, Moyano-López FJ, Civera-Cerecedo R, Carrasco-Chávez V, Ortiz-Galindo JL, Nolasco-Soria H, Tovar-Ramírez D, Dumas S (2010) Development of digestive enzyme activity in larvae of spotted sand bass Paralabrax maculatofasciatus II: electrophoretic analysis. Fish Physiol Biochem 36:29–37

    Article  PubMed  Google Scholar 

  • Benson D, Bogusk M, Lipman DJ, Ostell J (1994) Genbank. Nucleic Acids Res 22:3441–3444

    Article  PubMed  CAS  Google Scholar 

  • Bolognesi M, Gatti G, Menegatti E, Guarneri M, Marquart M, Pamakokos E, Huber R (1982) Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 Å resolution; structure solution, crystallographic refinement and preliminary structural interpretation. J Mol Biol 162:839–868

    Article  PubMed  CAS  Google Scholar 

  • Braun R, Arnesen JA, Rinne A, Hjelmeland K (1990) Immunohistological localization of trypsin in mucus-secreting cell layers of Atlantic salmon, Salmo salar L. J Fish Dis 13:233–238

    Article  Google Scholar 

  • Cahu C, Zambonino-Infante JL (1994) Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet: effect on digestive enzymes. Comp Biochem Physiol A 109:213–222

    Article  Google Scholar 

  • Chen JM, Kukor Z, Le Marechal C, Toth M, Taskiris L, Raguenes O, Ferec C, Sahin-Toth M (2003) Evolution of trypsinogen activation peptides. Mol Biol Evol 20:1767–1777

    Article  PubMed  CAS  Google Scholar 

  • Darias MJ, Murray HM, Gallant JW, Douglas SE, Yúfera M, Martínez-Rodríguez G (2007) The spatiotemporal expression pattern of trypsinogen and bile salt-activated lipase during the larval development of red porgy (Pagrus pagrus, Pisces, Sparidae). Mar Biol 152:109–118

    Article  CAS  Google Scholar 

  • Eusebio PS, Toledo JD, Mamauag REP, Bernas MJG (2004) Digestive enzyme activity in developing grouper (Epinephelus coioides) larvae. In: Rimmer MA, McBride S, Williams KC (eds) Advances in grouper aquaculture. Aust Center Int Agric Res, Canberra, pp 35–40

    Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: and approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Feng SZ, Li WS, Lin HR (2008) Identification and expression characterization of pepsinogen A in orange-spotted grouper, Epinephelus coioides. J Fish Biol 73:1960–1978

    Article  CAS  Google Scholar 

  • Fujii A, Kurokawa Y, Kawai S, Yoseda K, Dan S, Kai A, Tanaka M (2007) Diurnal variation of tryptic activity in larval stage and development of proteolytic enzyme activities of Malabar grouper (Epinephelus malabaricus) after hatching. Aquaculture 270:68–76

    Article  CAS  Google Scholar 

  • Galaviz MA, García-Ortega A, Gisbert E, López LM, Gasca AG (2012) Expression and activity of trypsin and pepsin during larval development of the spotted rose snapper Lutjanus guttatus. Comp Biochem Physiol B 161:9–16

    Article  PubMed  CAS  Google Scholar 

  • Gawlicka A, Parent B, Horn MH, Ross N, Opstad I, Torrinsen OJ (2000) Activity of digestive enzymes in yolksac larvae of Atlantic halibut (Hippoglossus hippoglossus): indication of readiness for first feeding. Aquaculture 184:303–314

    Article  CAS  Google Scholar 

  • Govoni JJ, Boehlert GW, Watanabe Y (1986) The physiology of digestion in fish larvae. Environ Biol Fish 16:59–77

    Article  Google Scholar 

  • Graf L, Jancso A, Szilagyi L, Hegyi G, Pinter K, Naray-Szabo G, Hepp J, Medzihradszky K, Rutter WJ (1988) Electrostatic complementarity within the substrate-binding pocket of trypsin. Proc Natl Acad Sci USA 85:4961–4965

    Article  PubMed  CAS  Google Scholar 

  • Gudmundsdottir A, Gudmundsdottir E, Oskarsson S, Bjarnason JB, Eakin AK, Craik CS (1993) Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen. Eur J Biochem 217:1091–1097

    Article  PubMed  CAS  Google Scholar 

  • Hedstrom L, Szilagyi L, Rutter WJ (1992) Converting trypsin to chymotrypsin: the role of surface loops. Science 255:1249–1253

    Article  PubMed  CAS  Google Scholar 

  • Hedstrom L, Perona JJ, Rutter WJ (1994) Converting trypsin to chymotrypsin: residue 172 is a substrate specificity determinant. Biochemistry 33:8757–8763

    Article  PubMed  CAS  Google Scholar 

  • Krem MM, Rose T, Cera ED (1999) The C-terminal sequence encodes function in serine proteases. J Biol Chem 274:28063–28066

    Article  PubMed  CAS  Google Scholar 

  • Kumar KJ, Tamura K, Nei M (1993) MEGA: molecular evolutionary genetics analysis, version 101. Pennsylvania State University, University Park

    Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Google Scholar 

  • Lazo JP, Holt GJ, Arnold CR (2000) Ontogeny of pancreatic enzymes in larval red drum Sciaenops ocellatus. Aquac Nutr 6:183–192

    Article  CAS  Google Scholar 

  • Lemieux H, Blier P, Dutil JD (1999) Do digestive enzymes set a physiological limit on growth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)? Fish Physiol Biochem 20:293–303

    Article  CAS  Google Scholar 

  • Light A, Janska H (1989) Enterokinase (enteropeptidase): comparative aspects. Trends Biochem Sci 14:110–112

    Article  PubMed  CAS  Google Scholar 

  • Lilleeng E, Froystand MK, Ostby GC, Valen EC, Krogdahl A (2007) Effects of diets containing soybean meal on trypsin mRNA expression and activity in Atlantic salmon (Salmo salar L). Comp Biochem Physiol A 147:25–36

    Article  Google Scholar 

  • Liu CH, Tseng MC, Cheng W (2007a) Identification and cloning of the antioxidant enzyme, glutathione peroxidase, of white shrimp, Litopenaeus vannamei, and its expression following Vibrio alginolyticus infection. Fish Shellfish Immunol 23:34–45

    Article  PubMed  Google Scholar 

  • Liu ZY, Wang Z, Xu SY, Xu LN (2007b) Two trypsin isoforms from the intestine of the grass carp (Ctenopharyngodon idellus). J Comp Physiol B 177:655–666

    Article  PubMed  CAS  Google Scholar 

  • Liu CH, Shiu YL, Hsu JL (2012) Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides. Fish Physiol Biochem 38:837–848

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Manchado M, Infante C, Asensio E, Crespo A, Zuasti E, Cañavate JP (2008) Molecular characterization and gene expression of six trypsinogens in the flatfish Senegalese sole (Solea senegalensis Kaup) during larval development and in tissues. Comp Biochem Physiol B 149:334–344

    Article  PubMed  Google Scholar 

  • Mithöfer K, Fernandez-del Castillo C, Rattner D, Warshaw AL (1998) Subcellular kinetics of early trypsinogen activation in acute rodent pancreatitis. Am J Physiol 274:G71–G79

    PubMed  Google Scholar 

  • Moyano FJ, Diaz M, Alarcon FJ, Sarasquete MC (1996) Characterization of digestive enzyme activity during larval development of gilthead sea bream (Sparus aurata). Fish Physiol Biochem 15:121–130

    Article  CAS  Google Scholar 

  • Murray HM, Perez-Casanova JC, Gallant JW, Johnson SC, Douglas SE (2004) Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus). Comp Biochem Physiol A 138:53–59

    CAS  Google Scholar 

  • Murray HM, Gallant JW, Johnson SC, Douglas SE (2006) Cloning and expression analysis of three digestive enzymes from Atlantic halibut (Hippoglossus hippoglossus) during early development: predicting gastrointestinal functionality. Aquaculture 252:394–408

    Article  CAS  Google Scholar 

  • Nesterov V, Dahlmann A, Bertog M, Korbmacher C (2008) Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron. Am J Physiol Renal Physiol 295:F1052–F1062

    Article  PubMed  CAS  Google Scholar 

  • Oozeki Y, Bailey KM (1995) Ontogenetic development of digestive enzyme activities in larval walleye Pollock, Theragra chalcogramma. Mar Biol 122:177–186

    CAS  Google Scholar 

  • Ostaszewska T, Korwin-Kossakowski M, Wolnicki J (2006) Morphological changes of digestive structures in starved tench Tinca tinca (L.) juveniles. Aquacult Int 14:113–126

    Article  Google Scholar 

  • Perez-Casanova JC, Murray HM, Gallant JW, Ross NW, Douglas SE, Johnson SC (2006) Development of the digestive capacity in larvae of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Aquaculture 251:377–401

    Article  Google Scholar 

  • Pierre S, Gaillard S, Prévot-D’alvise N, Aubert J, Rostaing-Capaillon O, Leung-Tack D, Grillasca JP (2008) Grouper aquaculture: Asian success and Mediterranean trials. Aquat Conserv Mar Freshw Ecosyst 18:297–308

    Article  Google Scholar 

  • Rawlings ND, Barrett AJ (1994) Families of serine peptidases. Meth Enzymol 244:19–61

    Article  PubMed  CAS  Google Scholar 

  • Roach JC (2002) A clade of trypsins found in cold-adapted fish. Proteins 47:31–44

    Article  PubMed  CAS  Google Scholar 

  • Roach JC, Wang K, Gan L, Hood L (1997) The molecular evolution of the vertebrate trypsinogens. J Mol Evol 45:640–652

    Article  PubMed  CAS  Google Scholar 

  • Ruan GL, Li Y, Gao ZX, Wang HL, Wang WM (2010) Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus). Comp Biochem Physiol B 155:77–85

    Article  PubMed  Google Scholar 

  • Rungruangsak-Torrissen K, Moss R, Andresen LH, Berg A, Waagbø R (2006) Different expressions of trypsin and chymotrypsin in relation to growth in Atlantic salmon (Salmon salar L.). Fish Physiol Biochem 32:7–23

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nat Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Zambonino-Infante JL, Cahu C (1994) Development and response to a diet change of some digestive enzyme in seabass (Dicentrarchus labrax) larvae. Fish Physiol Biochem 12:399–408

    Article  Google Scholar 

  • Zambonino-Infante JL, Cahu C (2001) Ontogeny of the gastrointestinal tract of marine fish larvae. Comp Biochem Physiol C 130:477–487

    CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by a grant from the National Science Council (NSC99-2313-B-020-005-MY3), Taiwan. The authors thank Sian-Ru Fu and Jhih-Syuan Chen who assisted with carrying out this project.

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  1. Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan, ROC

    Chun-Hung Liu, Ya-Huei Chen & Ya-Li Shiu

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  1. Chun-Hung Liu
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Correspondence to Chun-Hung Liu.

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Liu, CH., Chen, YH. & Shiu, YL. Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development. Fish Physiol Biochem 39, 201–214 (2013). https://doi.org/10.1007/s10695-012-9691-4

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