Plant Molecular Biology Reporter

, Volume 30, Issue 3, pp 624–632 | Cite as

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

  • Andrea Yamila Mansilla
  • Carmen Inés Segarra
  • Rubén Danilo Conde


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.


Germin-like protein Leaf apoplast Multifunctionality Serine protease inhibitor Wheat 



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.


  1. 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 PubMedCrossRefGoogle Scholar
  2. 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 CrossRefGoogle Scholar
  3. 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 CrossRefGoogle Scholar
  4. Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451PubMedCrossRefGoogle Scholar
  5. 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 342:221–224PubMedCrossRefGoogle Scholar
  6. Chaplin MF (1976) The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins. Biochem J 155:457–459PubMedGoogle Scholar
  7. 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 CrossRefGoogle Scholar
  8. 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
  9. 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 CrossRefGoogle Scholar
  10. 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 CrossRefGoogle Scholar
  11. Dunwell JM, Purvis A, Khuri S (2004) Cupins: the most functionally diverse protein superfamily? Phytochemistry 65:7–17PubMedCrossRefGoogle Scholar
  12. 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 PubMedCrossRefGoogle Scholar
  13. 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 12:564–573PubMedGoogle Scholar
  14. Garrels JI (1983) Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol 100:411–423PubMedCrossRefGoogle Scholar
  15. 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 2:669–678PubMedCrossRefGoogle Scholar
  16. 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 15:177–184PubMedCrossRefGoogle Scholar
  17. Hawkes R (1986) The dot immunobinding assay. Methods Enzymol 121:484–491PubMedCrossRefGoogle Scholar
  18. Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102:196–202PubMedCrossRefGoogle Scholar
  19. 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 22:937–952PubMedCrossRefGoogle Scholar
  20. Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. California Agricultural Experiment Station. Circular 347:1–32Google Scholar
  21. Huberts DHEW, van der Klei IJ (2010) Moonlighting proteins: An intriguing mode of multitasking. Biochim Biophys Acta (BBA) - Mol Cell Res 1803:520–525CrossRefGoogle Scholar
  22. Joubert FJ, Heussen C, Dowdle EB (1985) The complete amino acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds. J Biol Chem 260:12948–12953PubMedGoogle Scholar
  23. 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 97:2940–2945PubMedCrossRefGoogle Scholar
  24. 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 26:1601–1606PubMedCrossRefGoogle Scholar
  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  26. 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 1477:324–337CrossRefGoogle Scholar
  27. 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 CrossRefGoogle Scholar
  28. Mosolov VV, Valueva TA (2005) Proteinase inhibitors and their function in plants: a review. Prikladnaia biokhimiia i mikrobiologiia 41:261–282PubMedGoogle Scholar
  29. 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 43:1568–1574PubMedCrossRefGoogle Scholar
  30. 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 9:255–262PubMedCrossRefGoogle Scholar
  31. 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 PubMedCrossRefGoogle Scholar
  32. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567PubMedCrossRefGoogle Scholar
  33. 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 PubMedCrossRefGoogle Scholar
  34. Romaniouk A, Vijay IK (1997) Structure-function relationships in glucosidase I: amino acids involved in binding the substrate to the enzyme. Glycobiology 7:399–404PubMedCrossRefGoogle Scholar
  35. 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 150:105–111CrossRefGoogle Scholar
  36. 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 PubMedCrossRefGoogle Scholar
  37. 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 CrossRefGoogle Scholar
  38. 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 PubMedCrossRefGoogle Scholar
  39. 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 150:76–85PubMedCrossRefGoogle Scholar
  40. Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28(1102):1104Google Scholar
  41. 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 34:8281–8293PubMedCrossRefGoogle Scholar
  42. 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 CrossRefGoogle Scholar
  43. 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 255:5965–5970PubMedGoogle Scholar
  44. 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 37:297–308PubMedCrossRefGoogle Scholar
  45. 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 PubMedCrossRefGoogle Scholar
  46. 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
  47. 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 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Andrea Yamila Mansilla
    • 1
  • Carmen Inés Segarra
    • 1
    • 2
  • Rubén Danilo Conde
    • 1
  1. 1.Instituto de Investigaciones Biológicas (IIB), Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de Mar del Plata–CONICETMar del PlataArgentina
  2. 2.Departamento de Biología, Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de Mar del PlataMar del PlataArgentina

Personalised recommendations