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Structural and Functional Features of a Wheat Germin-Like Protein that Inhibits Trypsin

Abstract

The wheat leaf apoplast contains a protein that inhibits trypsin and belongs to the family of germin-like proteins called germin-like protease inhibitor (GLPI). Since it was first described in our laboratory, the objective of this study was to find out if GLPI is a new germin-like protein and to identify the molecular site responsible for its inhibitory action. Amino acid sequence fragments of GLPI have been determined using mass spectrometry and used to synthesize complementary DNA by reverse transcription PCR. This has allowed recovery of the amino acid sequence of the mature form of GLPI, which is indistinguishable from barley GLP and having pyrophosphatase/phosphodiesterase activity. Using chemical modifiers of amino acids, the unique Arg of GLPI is found to be necessary for preserving its protease inhibition activity. Furthermore, structural homology modeling has allowed prediction that Arg is located along the GLPI surface, which could aid in its activity on proteases. Given that GLPI acts as a superoxide dismutase and as pyrophosphatase/phosphodiesterase, it is deemed to be a multifunctional protein.

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References

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi:10.1093/bioinformatics/bti770

    PubMed  Article  CAS  Google Scholar 

  • Bernier F, Berna A (2001) Germins and germin-like proteins: plant do-all proteins. But what do they do exactly? Plant Physiol Biochem 39:545–554. doi:10.1016/s0981-9428(01)01285-2

    Article  CAS  Google Scholar 

  • Bian F, Zheng C, Qu F, Gong X, You C (2010) Proteomic analysis of somatic embryogenesis in Cyclamen persicum Mill. Plant Mol Biol Rep 28:22–31. doi:10.1007/s11105-009-0104-5

    Article  CAS  Google Scholar 

  • Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451

    PubMed  Article  CAS  Google Scholar 

  • Ceciliani F, Bortolotti F, Menegatti E, Ronchi S, Ascenzi P, Palmieri S (1994) Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (Brassica napus) seed. FEBS Lett 342:221–224

    PubMed  Article  CAS  Google Scholar 

  • Chaplin MF (1976) The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins. Biochem J 155:457–459

    PubMed  CAS  Google Scholar 

  • Chen N, Liu Y, Liu X, Chai J, Hu Z, Guo G, Liu H (2009) Enhanced tolerance to water deficit and salinity stress in transgenic Lycium barbarum L. plants ectopically expressing athk1, an Arabidopsis thaliana histidine kinase gene. Plant Mol Biol Rep 27:321–333. doi:10.1007/s11105-008-0084-x

    Article  Google Scholar 

  • Chen X, Wang M, Holbrook C, Culbreath A, Liang X, Brenneman T, Guo B (2011) Identification and Characterization of a Multigene Family Encoding Germin-Like Proteins in Cultivated Peanut (Arachis hypogaea L.). Plant Mol Biol Rep. doi:10.1007/s11105-010-0237-6

  • Cordo CA, Monaco CI, Segarra CI, Simon MR, Mansilla AY, Perelló AE, Kripelz NI, Bayo D, Conde RD (2007) Trichoderma spp. as elicitors of wheat plant defense responses against Septoria tritici. Biocontrol Sci Technol 17:687–698. doi:10.1080/09583150701527094

    Article  Google Scholar 

  • Davidson RM, Reeves PA, Manosalva PM, Leach JE (2009) Germins: A diverse protein family important for crop improvement. Plant Sci 177:499–510. doi:10.1016/j.plantsci.2009.08.012

    Article  CAS  Google Scholar 

  • Dunwell JM, Purvis A, Khuri S (2004) Cupins: the most functionally diverse protein superfamily? Phytochemistry 65:7–17

    PubMed  Article  CAS  Google Scholar 

  • El-Sharkawy I, Mila I, Bouzayen M, Jayasankar S (2010) Regulation of two germin-like protein genes during plum fruit development. J Exp Bot 61:1761–1770. doi:10.1093/jxb/erq043

    PubMed  Article  CAS  Google Scholar 

  • Fernández-Patrón C, Castellanos-Serra L, Rodriguez P (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins. Biotechniques 12:564–573

    PubMed  Google Scholar 

  • Garrels JI (1983) Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol 100:411–423

    PubMed  Article  CAS  Google Scholar 

  • Grasberger BL, Clore GM, Gronenborn AM (1994) High-resolution structure of Ascaris trypsin inhibitor in solution: direct evidence for a pH-induced conformational transition in the reactive site. Structure 2:669–678

    PubMed  Article  CAS  Google Scholar 

  • Haldar UC, Saha SK, Beavis RC, Sinha NK (1996) Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: purification, properties, and amino acid sequences. J Protein Chem 15:177–184

    PubMed  Article  CAS  Google Scholar 

  • Hawkes R (1986) The dot immunobinding assay. Methods Enzymol 121:484–491

    PubMed  Article  CAS  Google Scholar 

  • Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102:196–202

    PubMed  Article  CAS  Google Scholar 

  • Himmelbach A, Liu L, Zierold U, Altschmied L, Maucher H, Beier F, Müller D, Hensel G, Heise A, Schützendübel A, Kumlehn J, Schweizer P (2010) Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack. Plant Cell 22:937–952

    PubMed  Article  CAS  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. California Agricultural Experiment Station. Circular 347:1–32

    Google Scholar 

  • Huberts DHEW, van der Klei IJ (2010) Moonlighting proteins: An intriguing mode of multitasking. Biochim Biophys Acta (BBA) - Mol Cell Res 1803:520–525

    Article  CAS  Google Scholar 

  • Joubert FJ, Heussen C, Dowdle EB (1985) The complete amino acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds. J Biol Chem 260:12948–12953

    PubMed  CAS  Google Scholar 

  • Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci U S A 97:2940–2945

    PubMed  Article  CAS  Google Scholar 

  • Kumar RS, Suresh CG, Pundle A, Prabhune A (2004) Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila. Biotechnol Lett 26:1601–1606

    PubMed  Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Article  CAS  Google Scholar 

  • Laskowski M, Qasim MA (2000) What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim Biophys Acta (BBA) - Protein Struct Mol Enzymol 1477:324–337

    Article  CAS  Google Scholar 

  • Marcus J, Goulter K, Manners J (2008) Peptide Fragments From Plant Vicilins Expressed in Escherichia Coli Exhibit Antimicrobial Activity In Vitro. Plant Mol Biol Rep 26:75–87. doi:10.1007/s11105-008-0024-9

    Article  CAS  Google Scholar 

  • Mosolov VV, Valueva TA (2005) Proteinase inhibitors and their function in plants: a review. Prikladnaia biokhimiia i mikrobiologiia 41:261–282

    PubMed  CAS  Google Scholar 

  • Nakata M, Shiono T, Watanabe Y, Satoh T (2002) Salt stress-induced dissociation from cells of a germin-like protein with Mn-SOD activity and an increase in its mRNA in a moss, Barbula unguiculata. Plant Cell Physiol 43:1568–1574

    PubMed  Article  CAS  Google Scholar 

  • Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262

    PubMed  Article  CAS  Google Scholar 

  • Opaleye O, Rose RS, Whittaker MM, Woo EJ, Whittaker JW, Pickersgill RW (2006) Structural and spectroscopic studies shed light on the mechanism of oxalate oxidase. J Biol Chem 281:6428–6433. doi:10.1074/jbc.M510256200

    PubMed  Article  CAS  Google Scholar 

  • Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    PubMed  Article  CAS  Google Scholar 

  • Rodríguez-López M, Baroja-Fernandez E, Zandueta-Criado A, Moreno-Bruna B, Munoz FJ, Akazawa T, Pozueta-Romero J (2001) Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein. FEBS Lett 490:44–48. doi:10.1016/S0014-5793(01)02135-4

    PubMed  Article  Google Scholar 

  • Romaniouk A, Vijay IK (1997) Structure-function relationships in glucosidase I: amino acids involved in binding the substrate to the enzyme. Glycobiology 7:399–404

    PubMed  Article  CAS  Google Scholar 

  • Segarra CI, Casalongué CA, Pinedo ML, Cordo CA, Conde RD (2002) Changes in Wheat Leaf Extracellular Proteolytic Activity after Infection with Septoria tritici. J Phytopathol 150:105–111

    Article  Google Scholar 

  • Segarra CI, Casalongué CA, Pinedo ML, Ronchi VP, Conde RD (2003) A germin-like protein of wheat leaf apoplast inhibits serine proteases. J Exp Bot 54:1335–1341. doi:10.1093/jxb/erg139

    PubMed  Article  CAS  Google Scholar 

  • Shetty N, Jørgensen H, Jensen J, Collinge D, Shetty H (2008) Roles of reactive oxygen species in interactions between plants and pathogens. Eur J Plant Pathol 121:267–280. doi:10.1007/s10658-008-9302-5

    Article  CAS  Google Scholar 

  • Shetty NP, Jensen JD, Knudsen A, Finnie C, Geshi N, Blennow A, Collinge DB, Jorgensen HJ (2009) Effects of beta-1,3-glucan from Septoria tritici on structural defence responses in wheat. J Exp Bot 60:4287–4300. doi:10.1093/jxb/erp269

    PubMed  Article  CAS  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    PubMed  Article  CAS  Google Scholar 

  • Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28(1102):1104

    Google Scholar 

  • Strobl S, Muhlhahn P, Bernstein R, Wiltscheck R, Maskos K, Wunderlich M, Huber R, Glockshuber R, Holak TA (1995) Determination of the three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds by NMR spectroscopy. Biochemistry 34:8281–8293

    PubMed  Article  CAS  Google Scholar 

  • Tabuchi T, Kumon T, Azuma T, Nanmori T, Yasuda T (2003) The expression of a germin-like protein with superoxide dismutase activity in the halophyte Atriplex lentiformis is differentially regulated by wounding and abscisic acid. Physiol Plant 118:523–531. doi:10.1034/j.1399-3054.2003.00133.x

    Article  CAS  Google Scholar 

  • Thompson EW, Lane BG (1980) Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk mRNA from dry and imbibing embryos. J Biol Chem 255:5965–5970

    PubMed  CAS  Google Scholar 

  • Vallelian-Bindschedler L, Mosinger E, Metraux JP, Schweizer P (1998) Structure, expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leaves. Plant Mol Biol 37:297–308

    PubMed  Article  CAS  Google Scholar 

  • Woo EJ, Dunwell JM, Goodenough PW, Marvier AC, Pickersgill RW (2000) Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities. Nat Struct Biol 7:1036–1040. doi:10.1038/80954

    PubMed  Article  CAS  Google Scholar 

  • Xu C, Zheng L, Gao C, Wang C, Liu G, Jiang J, Wang Y (2011) Ovexpression of a Vacuolar H + −ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco. Plant Mol Biol Rep. doi:10.1007/s11105-010-0247-4

  • Zimmermann G, Baumlein H, Mock HP, Himmelbach A, Schweizer P (2006) The multigene family encoding germin-like proteins of barley. Regulation and function in Basal host resistance. Plant Physiol 142:181–192. doi:10.1104/pp.106.083824

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

Grants to RD Conde of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Universidad Nacional de Mar del Plata (UNMdP) supported this work. AY Mansilla is a doctoral student funded by CONICET.

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Correspondence to Andrea Yamila Mansilla.

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Mansilla, A.Y., Segarra, C.I. & Conde, R.D. Structural and Functional Features of a Wheat Germin-Like Protein that Inhibits Trypsin. Plant Mol Biol Rep 30, 624–632 (2012). https://doi.org/10.1007/s11105-011-0372-8

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  • DOI: https://doi.org/10.1007/s11105-011-0372-8

Keywords

  • Germin-like protein
  • Leaf apoplast
  • Multifunctionality
  • Serine protease inhibitor
  • Wheat