Journal of Molecular Evolution

, Volume 59, Issue 4, pp 437–447 | Cite as

Serpins in Unicellular Eukarya, Archaea, and Bacteria: Sequence Analysis and Evolution

  • Thomas H. Roberts
  • Jørn Hejgaard
  • Neil F. W. Saunders
  • Ricardo Cavicchioli
  • Paul M. G. Curmi


Most serpins irreversibly inactivate specific serine proteinases of the chymotrypsin family. Inhibitory serpins are unusual proteins in that their native structure is metastable, and rapid conversion to a relaxed state is required to trap target enzymes in a covalent complex. The evolutionary origin of the serpin fold is unresolved, and while serpins in animals are known to be involved in the regulation of a remarkable diversity of metabolic processes, the physiological functions of homologues from other phyla are unknown. Addressing these questions, here we analyze serpin genes identified in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea, where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics of inhibitory serpins, and where multiple serpin genes are found in one genome, variability is displayed in the region of the reactive-center loop important for specificity. All the unicellular eukaryotic serpins have large hydrophobic or positively charged residues at the putative P1 position. In contrast, none of the prokaryotic serpins has a residue of these types at the predicted P1 position, but many have smaller, neutral residues. Serpin evolution is discussed.


Genome Phylogeny Prokaryote Protein fold Reactive-center loop Green alga Codon usage Protostome–deuterostome split Proteinase Protease 



We thank Stephen Craft for technical editing of the manuscript, Nishen Naidoo for assistance in production of Fig. 1, and David Briscoe for helpful discussions. Research by N.F.W.S., R.C., and P.M.G.C. is supported by the Australian Research Council.


  1. Altschul, SF, Madden, TL, Schäffer, AA, Zhang, J, Zhang, Z, Miller, W, Lipman, DJ 1997Gapped BLAST and PSI-BLAST: A new generation of protein database search programsNucleic Acids Res2533893402PubMedGoogle Scholar
  2. Atchley, WR, Lokot, T, Wollenberg, K, Dress, A, Ragg, H 2001Phylogenetic analyses of amino acid variation in the serpin proteinsMol Biol Evol1815021511PubMedGoogle Scholar
  3. Bao, Q, Tian, Y, Li, W, Xu, Z, Xuan, Z, Hu, S, Dong, W, Yang, J, Chen, Y, Xue, Y, Xu, Y, Lai, X, Huang, L, Dong, X, Ma, Y, Ling, L, Tan, H, Chen, R, Wang, J, Yu, H, Yang, J 2002A complete sequence of the T. tengcongensis genomeGenome Res12689700CrossRefPubMedGoogle Scholar
  4. Barbour, KW, Goodwin, RL, Guillonneau, F, Wang, Y, Baumann, H, Berger, FG 2002Functional diversification during evolution of the murine alpha(1)-proteinase inhibitor family: Role of the hypervariable reactive center loopMol Biol Evol19718727PubMedGoogle Scholar
  5. Bollen, A, Herzog, A, Cravador, A, Herion, P, Chuchana, P, Vander-Straten, A, Loriau, R, Jacobs, P, Elsen, A 1983Cloning and expression in Escherichia coli of full-length complementary DNA coding for human alpha 1-antitrypsinDNA2255264PubMedGoogle Scholar
  6. Bonaldo, MF, Lennon, G, Soares, MB 1996Normalization and subtraction: Two approaches to facilitate gene discoveryGenome Res6791806PubMedGoogle Scholar
  7. Boone, DRCastenholz, GMGarrity, GM eds. 2001Bergey’s manual of systematic bacteriology (2nd ed.), Vol 1SpringerNew YorkGoogle Scholar
  8. Carrell, RW, Travis, J 1985Alpha-1-antitrypsin and the serpins: variation and countervariationTrends Biochem Sci102024CrossRefGoogle Scholar
  9. Dahlen, JR, Foster, DC, Kisiel, W 1997Human proteinase inhibitor 9 (PI9) is a potent inhibitor of subtilisinBiochem Biophys Res Commun238329333CrossRefPubMedGoogle Scholar
  10. DeLano, WL 2002The PyMOL molecular graphics systemDeLano ScientificSan Carlos, CAGoogle Scholar
  11. Deppenmeier, U, Johann, A, Hartsch, T, Merkl, R, Schmitz, RA, Martinez-Arias, R, Henne, A, Wiezer, A, Baeumer, S, Jacobi, C, Brueggemann, H, Lienard, T, Christmann, A, Boemecke, M, Steckel, S, Bhattacharyya, A, Lykidis, A, Overbeek, R, Klenk, H-P, Gunsalus, RP, Fritz, H-J, Gottschalk, G 2002The genome of Methanosarcina mazei: Evidence for lateral gene transfer between Bacteria and ArchaeaJ Mol Microbiol Biotechnol4453461PubMedGoogle Scholar
  12. Elliott, PR, Abrahams, JP, Lomas, DA 1998Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformationJ Mol Biol275419425CrossRefPubMedGoogle Scholar
  13. Faust, MA, Gulledge, RA 2002Identifying harmful marine dinoflagellatesContrib US Nat Herb421144Google Scholar
  14. Fitz-Gibbon, ST, Ladner, H, Kim, UJ, Stetter, KO, Simon, MI, Miller, JH 2002Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilumProc Natl Acad Sci USA99984989CrossRefPubMedGoogle Scholar
  15. Gettins, PGW 2002Serpin structure, mechanism and functionChem Rev10247514803CrossRefPubMedGoogle Scholar
  16. Harris, EH 1989The Chlamydomonas sourcebook: A comprehensive guide to biology and laboratory useAcademic PressLondonGoogle Scholar
  17. Harrop, SJ, Jankova, L, Coles, M, Jardine, D, Whittaker, JS, Gould, AR, Meister, A, King, GC, Mabbutt, BC, Curmi, PM 1999The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin functionStruct Fold Des74354CrossRefGoogle Scholar
  18. Hejgaard, J, Hauge, S 2002Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificityPhysiol Plant116155163CrossRefPubMedGoogle Scholar
  19. Hill, RE, Hastie, ND 1987Accelerated evolution in the reactive centre regions of serine protease inhibitorsNature3269699CrossRefPubMedGoogle Scholar
  20. Hopkins, PC, Carrell, RW, Stone, SR 1993Effects of mutations in the hinge region of serpinsBiochemistry3276507657PubMedGoogle Scholar
  21. Huber, R, Carrell, RW 1989Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpinsBiochemistry2889518966PubMedGoogle Scholar
  22. Huntington, JA, Read, RJ, Carrell, RW 2000Structure of a serpin-protease complex shows inhibition by deformationNature407923926CrossRefPubMedGoogle Scholar
  23. Irving, JA, Pike, RN, Lesk, AM, Whisstock, JC 2000Phylogeny of the serpin superfamily: Implications of patterns of amino acid conservation for structure and functionGenome Res1018451864CrossRefPubMedGoogle Scholar
  24. Irving, JA, Steenbakkers, PJM, Lesk, AM, Op den Camp, HJM, Pike, RN, Whisstock, JC 2002Serpins in prokaryotesMol Biol Evol1918811890PubMedGoogle Scholar
  25. Irving, JA, Cabrita, LD, Rossjohn, J, Pike, RN, Bottomley, SP, Whisstock, JC 2003The 1.5Å crystal structure of a prokaryote serpin: Controlling conformational change in a heated environmentStructure11387397CrossRefPubMedGoogle Scholar
  26. Jiang, H, Wang, Y, Huang, Y, Mulnix, AB, Kadel, J, Cole, K, Kanost, MR 1996Organization of serpin gene-1 from Mandura sextaJ Biol Chem2712801728023CrossRefPubMedGoogle Scholar
  27. Kaneko, T, Nakamura, Y, Wolk, CP, Kuritz, T, Sasamoto, S, Watanabe, A, Iriguchi, M, Ishikawa, A, Kawashima, K, Kimura, T, Kishida, Y, Kohara, M, Matsumoto, M, Matsuno, A, Muraki, A, Nakazaki, N, Shimpo, S, Sugimoto, M, Takazawa, M, Yamada, M, Yasuda, M, Tabata, S 2001Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120DNA Res8205213PubMedGoogle Scholar
  28. Keates, SE, Kostman, TA, Anderson, JD, Bailey, BA 2003Altered gene expression in three plant species in response to treatment with Nep1, a fungal protein that causes necrosisPlant Physiol13216101622CrossRefPubMedGoogle Scholar
  29. Krem, MM, Di Cera, E 2003Conserved Ser residues, the shutter region, and speciation in serpin evolutionJ Biol Chem2783781037814CrossRefPubMedGoogle Scholar
  30. Kruger, O, Ladewig, J, Koster, K, Ragg, H 2002Widespread occurrence of serpin genes with multiple reactive centre-containing exon cassettes in insects and nematodesGene29397105CrossRefPubMedGoogle Scholar
  31. Loebermann, H, Tokuoka, R, Deisenhofer, J, Huber, R 1984Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for functionJ Mol Biol177531557CrossRefPubMedGoogle Scholar
  32. Lomas, DA, Evans, DL, Upton, C, McFadden, G, Carrell, RW 1993Inhibition of plasmin, urokinase, tissue plasminogen activator, and CIS by a myxoma virus serine proteinase inhibitorJ Biol Chem268516521PubMedGoogle Scholar
  33. Lu, J, Nogi, Y, Takami, H 2001Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya RidgeFEMS Microbiol Lett205291297CrossRefPubMedGoogle Scholar
  34. Østergaard, H, Rasmussen, SK, Roberts, TH, Hejgaard, J 2000Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular formsJ Biol Chem2753327233279CrossRefPubMedGoogle Scholar
  35. Ray, CA, Black, RA, Kronheim, SR, Greenstreet, TA, Sleath, PR, Salvesen, GS, Pickup, DJ 1992Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzymeCell69597604CrossRefPubMedGoogle Scholar
  36. Renatus, M, Zhou, Q, Stennicke, HR, Snipas, SJ, Turk, D, Bankston, LA, Liddington, RC, Salvesen, GS 2000Crystal structure of the apoptotic suppressor CrmA in its cleaved formStruct Fold Des8789797CrossRefGoogle Scholar
  37. Riahi, Y, Siman-Tov, R, Ankri, S 2004Molecular cloning, expression and characterization of a serine protease inhibitor gene from Entamoeba histolyticaMol Biochem Parasitol133153162CrossRefPubMedGoogle Scholar
  38. Roberts, TH, Marttila, S, Rasmussen, SK, Hejgaard, J 2003Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barleyJ Exp Bot5422512263CrossRefPubMedGoogle Scholar
  39. Rosenkrands, I, Hejgaard, J, Rasmussen, SK, Bjorn, SE 1994Serpins from wheat grainFEBS Lett3437580CrossRefPubMedGoogle Scholar
  40. Rutherford, K, Parkhill, J, Crook, J, Horsnell, T, Rice, P, Rajandream, M-A, Barrell, B 2000Artemis: sequence visualisation and annotationBioinformatics16944945CrossRefPubMedGoogle Scholar
  41. Saunders, DN, Jankova, L, Harrop, SJ, Curmi, PM, Gould, AR, Ranson, M, Baker, MS 2001Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2J Biol Chem2764338343389CrossRefPubMedGoogle Scholar
  42. Saunders, NFW, Thomas, T, Curmi, PMG, Mattick, JS, Kuczek, E, Slade, R, Davis, J, Franzmann, PD, Boone, D, Rusterholtz, K, Feldman, R, Gates, C, Bench, S, Sowers, K, Kadner, K, Aerts, A, Dehal, P, Detter, C, Glavina, T, Lucas, S, Richardson, P, Larimer, F, Hauser, L, Land, M, Cavicchioli, R 2003Mechanisms of thermal adaptation revealed from the genomes of the Antarctic Archaea, Methanogenium frigidum and Methanococcoides burtoniiGenome Res1315801588CrossRefPubMedGoogle Scholar
  43. Schell, MA, Karmirantzou, M, Snel, B, Vilanova, D, Berger, B, Pessi, G, Zwahlen, MC, Desiere, F, Bork, P, Delley, M, Pridmore, RD, Arigoni, F 2002The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tractProc Natl Acad Sci USA991442214427CrossRefPubMedGoogle Scholar
  44. Schick, C, Pemberton, PA, Shi, GP, Kamachi, Y, Cataltepe, S, Bartuski, AJ, Gornstein, ER, Bromme, D, Chapman, HA, Silverman, GA 1998Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: A kinetic analysisBiochemistry3752585266CrossRefPubMedGoogle Scholar
  45. Silverman, GA, Bird, PI, Carrell, RW, Church, FC, Coughlin, PB, Gettins, PGW, Irving, JA, Lomas, DA, Luke, CJ, Moyer, RW, Pemberton, PA, Remold-O’Donnell, E, Salvesen, GS, Travis, J, Whisstock, JC 2001The serpins are an expanding superfamily of structurally similar but functionally diverse proteins: Evolution, mechanism of inhibition, novel functions, and a revised nomenclatureJ Biol Chem2763329333296CrossRefPubMedGoogle Scholar
  46. Simonovic, M, Gettins, PGW, Volz, K 2000Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibitionProtein Sci914231427PubMedGoogle Scholar
  47. Stein, PE, Carrell, RW 1995What do dysfunctional serpins tell us about molecular mobility and disease? Nat Struct Biol296113CrossRefPubMedGoogle Scholar
  48. Stratikos, E, Gettins, PG 1999Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet AProc Natl Acad Sci USA9648084813CrossRefPubMedGoogle Scholar
  49. Takami, H, Takaki, Y, Uchiyama, I 2002Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environmentsNucleic Acids Res3039273935CrossRefPubMedGoogle Scholar
  50. Thompson, JD, Gibson, TJ, Plewniak, F, Jeanmougin, F, Higgins, DG 1997The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis toolsNucleic Acids Res2448764882CrossRefGoogle Scholar
  51. Whisstock, JC, Skinner, R, Carrell, RW, Lesk, AM 2000Conformational changes in serpins: I. The native and cleaved conformations of α1-antitrypsinJ Mol Biol1651665CrossRefGoogle Scholar
  52. Ye, S, Cech, AL, Belmares, R, Bergstrom, RC, Tong, Y, Corey, DR, Kanost, MR, Goldsmith, EJ 2001The structure of a Michaelis serpin-protease complexNat Struct Biol8979983CrossRefPubMedGoogle Scholar
  53. Yoo, BC, Aoki, K, Xiang, Y, Campbell, LR, Hull, RJ, Xoconostle-Cazares, B, Monzer, J, Lee, JY, Ullman, DE, Lucas, WJ 2000Characterization of Cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitorJ Biol Chem2753512235128CrossRefPubMedGoogle Scholar
  54. Zang, X, Maizels, RM 2001Serine protease inhibitors from nematodes and the arms race between host and pathogenTrends Biochem Sci26191197CrossRefPubMedGoogle Scholar
  55. Zhou, A, Carrell, RW, Huntington, JA 2001The serpin inhibitory mechanism is critically dependent on the length of the reactive center loopJ Biol Chem292754 l27547Google Scholar

Copyright information

© Springer 2004

Authors and Affiliations

  • Thomas H. Roberts
    • 1
  • Jørn Hejgaard
    • 2
  • Neil F. W. Saunders
    • 3
  • Ricardo Cavicchioli
    • 3
  • Paul M. G. Curmi
    • 4
    • 5
  1. 1.Department of Biological SciencesMacquarie UniversityAustralia
  2. 2.Section of Biochemistry and Nutrition, BioCentrum-DTUTechnical University of DenmarkLyngbyDenmark
  3. 3.School of Biotechnology and Biomolecular SciencesUniversity of New South WalesAustralia
  4. 4.School of PhysicsUniversity of New South WalesAustralia
  5. 5.Centre for ImmunologySt.Vincent’s HospitalDarlinghurstAustralia

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