Abstract
The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein–protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- TG2:
-
Transglutaminase 2
- PU:
-
Putrescine
- SPD:
-
Spermidine
- SPM:
-
Spermine
- GA:
-
Glutaraldehyde
- BSA:
-
Bovine serum albumin
- OVA:
-
Ovalbumin
- HSP70:
-
Heat shock protein 70
- HSP90:
-
Heat shock protein 90
- GP:
-
Guinea pig
- ACTB:
-
Beta actin
- TUBA1A:
-
Alpha tubulin 1a
- TUBB:
-
Beta tubulin
- DTT:
-
Dithiothreitol
- DNC:
-
Monodansylcadaverine
- PBS:
-
Phosphate-buffered saline
- TBS:
-
Tris-buffered saline
References
Ahern GP, Wang X, Miyares RL (2006) Polyamines are potent ligands for the capsaicin receptor TRPV1. J Biol Chem 281:8991–8995. doi:10.1074/jbc.M513429200
Anderson PJ, Bardocz S, Campos R, Brown DL (1985) The effect of polyamines on tubulin assembly. Biochem Biophys Res Commun 132:147–154
Antonyak MA, Li B, Regan AD, Feng Q, Dusaban SS, Cerione RA (2009) Tissue transglutaminase is an essential participant in the epidermal growth factor-stimulated signaling pathway leading to cancer cell migration and invasion. J Biol Chem 284:17914–17925. doi:10.1074/jbc.M109.013037
Ballestar E, Abad C, Franco L (1996) Core histones are glutaminyl substrates for tissue transglutaminase. J Biol Chem 271:18817–18824
Boquet P (2001) The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. Toxicon: Off J Int Soc Toxinol 39:1673–1680
Boroughs LK, Antonyak MA, Johnson JL, Cerione RA (2011) A unique role for heat shock protein 70 and its binding partner tissue transglutaminase in cancer cell migration. J Biol Chem 286:37094–37107. doi:10.1074/jbc.M111.242438
Caprioli A et al (1987) Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections. J Clin Microbiol 25:146–149
Cason AL et al (2003) X-linked spermine synthase gene (SMS) defect: the first polyamine deficiency syndrome. EJHG 11:937–944. doi:10.1038/sj.ejhg.5201072
Cocuzzi E, Piacentini M, Beninati S, Chung SI (1990) Post-translational modification of apolipoprotein B by transglutaminases. Biochem J 265:707–713
Cooper HL, Park MH, Folk JE, Safer B, Braverman R (1983) Identification of the hypusine-containing protein hy+ as translation initiation factor eIF-4D. Proc Natl Acad Sci USA 80:1854–1857
Cooper AJ, Wang J, Pasternack R, Fuchsbauer HL, Sheu RK, Blass JP (2000) Lysine-rich histone (H1) is a lysyl substrate of tissue transglutaminase: possible involvement of transglutaminase in the formation of nuclear aggregates in (CAG)(n)/Q(n) expansion diseases. Dev Neurosci 22:404–417. doi:10.1159/000017470
Cordella-Miele E, Miele L, Beninati S, Mukherjee AB (1993) Transglutaminase-catalyzed incorporation of polyamines into phospholipase A2. J Biochem 113:164–173
Del Duca S, Serafini-Fracassini D, Bonner P, Cresti M, Cai G (2009) Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments. Biochem J 418:651–664. doi:10.1042/BJ20081781
D’Eletto M et al (2009) Transglutaminase 2 is involved in autophagosome maturation. Autophagy 5:1145–1154
Estepa I, Pestana A (1981) Activation by polyamines of the acetylation of endogenous histones in isolated chromatin and nuclei from Artemia. Eur J Biochem/FEBS 119:431–436
Folk JE (1980) Transglutaminases. Annu Rev Biochem 49:517–531. doi:10.1146/annurev.bi.49.070180.002505
Folk JE, Chung SI (1985) Transglutaminases. Methods Enzymol 113:358–375
Folk JE, Park MH, Chung SI, Schrode J, Lester EP, Cooper HL (1980) Polyamines as physiological substrates for transglutaminases. J Biol Chem 255:3695–3700
Fujiwara K (1994) An evaluation of polyamine immunocytochemistry using immunocytochemical model systems. Histochemistry 101:287–294
Fujiwara K, Araki M, Kitagawa T, Inoue Y (1993) A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model. Histochemistry 99:477–483
Gallo CJ, Koza RA, Herbst EJ (1986) Polyamines and HeLa-cell DNA replication. Biochem J 238:37–42
Goyns MH (1980) Effect of cell synchronization techniques on polyamine content of HeLa cells. Experientia 36:936–937
Gundemir S, Colak G, Tucholski J, Johnson GV (2012) Transglutaminase 2: a molecular Swiss army knife. Biochim Biophys Acta 1823:406–419. doi:10.1016/j.bbamcr.2011.09.012
Hamon L, Savarin P, Curmi PA, Pastre D (2011) Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines. Biophys J 101:205–216. doi:10.1016/j.bpj.2011.05.022
Hetrick B, Khade PK, Lee K, Stephen J, Thomas A, Joseph S (2010) Polyamines accelerate codon recognition by transfer RNAs on the ribosome. Biochemistry 49:7179–7189. doi:10.1021/bi1009776
Horiguchi Y, Inoue N, Masuda M, Kashimoto T, Katahira J, Sugimoto N, Matsuda M (1997) Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho. Proc Natl Acad Sci USA 94:11623–11626
Ientile R, Caccamo D, Griffin M (2007) Tissue transglutaminase and the stress response. Amino Acids 33:385–394. doi:10.1007/s00726-007-0517-0
Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol 42:39–51. doi:10.1016/j.biocel.2009.07.009
Jeitner TM, Matson WR, Folk JE, Blass JP, Cooper AJ (2008) Increased levels of gamma-glutamylamines in Huntington disease CSF. J Neurochem 106:37–44. doi:10.1111/j.1471-4159.2008.05350.x
Jeitner TM, Pinto JT, Krasnikov BF, Horswill M, Cooper AJ (2009) Transglutaminases and neurodegeneration. J Neurochem 109(Suppl 1):160–166. doi:10.1111/j.1471-4159.2009.05843.x
Jeitner TM, Battaile K, Cooper AJ (2013) Gamma-glutamylamines and neurodegenerative diseases. Amino Acids 44:129–142. doi:10.1007/s00726-011-1209-3
Jeon JH et al (2003a) Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine. EMBO J 22:5273–5282. doi:10.1093/emboj/cdg495
Jeon JH et al (2003b) Differential incorporation of biotinylated polyamines by transglutaminase 2. FEBS Lett 534:180–184
Jeon JH et al (2006) Alteration of Rb binding to HPV 18 E7 modified by transglutaminase 2 with different type of polyamines. Front Biosci: J Virtual Libr 11:1540–1548
Keillor JW, Clouthier CM, Apperley KY, Akbar A, Mulani A (2014) Acyl transfer mechanisms of tissue transglutaminase. Bioorg Chem. doi:10.1016/j.bioorg.2014.06.003
Kim JH, Nam KH, Kwon OS, Kim IG, Bustin M, Choy HE, Park SC (2002) Histone cross-linking by transglutaminase. Biochem Biophys Res Commun 293:1453–1457. doi:10.1016/S0006-291X(02)00393-5
Korolev N, Allahverdi A, Yang Y, Fan Y, Lyubartsev AP, Nordenskiold L (2010) Electrostatic origin of salt-induced nucleosome array compaction. Biophys J 99:1896–1905. doi:10.1016/j.bpj.2010.07.017
Lee DY, Chang GD (2014) Methylglyoxal in cells elicits a negative feedback loop entailing transglutaminase 2 and glyoxalase 1. Redox Biol 2:196–205. doi:10.1016/j.redox.2013.12.024
Lefevre J et al (2011) The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium. J Biol Chem 286:3065–3078. doi:10.1074/jbc.M110.144089
Lentini A, Tabolacci C, Melino S, Provenzano B, Beninati S (2010) Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity. Biochem Biophys Res Commun 393:546–550. doi:10.1016/j.bbrc.2010.02.060
Lorenz B et al (1998) Spermine deficiency in Gy mice caused by deletion of the spermine synthase gene. Hum Mol Genet 7:541–547
Makitie LT, Kanerva K, Andersson LC (2009) Ornithine decarboxylase regulates the activity and localization of rhoA via polyamination. Exp Cell Res 315:1008–1014. doi:10.1016/j.yexcr.2009.01.029
Masuda M, Betancourt L, Matsuzawa T, Kashimoto T, Takao T, Shimonishi Y, Horiguchi Y (2000) Activation of rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin. EMBO J 19:521–530. doi:10.1093/emboj/19.4.521
McConoughey SJ, Niatsetskaya ZV, Pasternack R, Hils M, Ratan RR, Cooper AJ (2009) A nonradioactive dot blot assay for transglutaminase activity. Anal Biochem 390:91–93. doi:10.1016/j.ab.2009.03.022
McCormack SA, Wang JY, Johnson LR (1994) Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells. Am J Physiol 267:C715–C722
Mechulam A, Chernov KG, Mucher E, Hamon L, Curmi PA, Pastre D (2009) Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion. PLoS Comput Biol 5:e1000255. doi:10.1371/journal.pcbi.1000255
Meyer RA Jr, Henley CM, Meyer MH, Morgan PL, McDonald AG, Mills C, Price DK (1998) Partial deletion of both the spermine synthase gene and the Pex gene in the X-linked hypophosphatemic, gyro (Gy) mouse. Genomics 48:289–295. doi:10.1006/geno.1997.5169
Mishra S, Saleh A, Espino PS, Davie JR, Murphy LJ (2006) Phosphorylation of histones by tissue transglutaminase. J Biol Chem 281:5532–5538. doi:10.1074/jbc.M506864200
Morselli E et al (2009) Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol. Aging 1:961–970
Muhlrad A, Grintsevich EE, Reisler E (2011) Polycation induced actin bundles. Biophys Chem 155:45–51. doi:10.1016/j.bpc.2011.02.008
Murakami K, Haneda M, Iwata S, Yoshino M (2012) Differential effects of polyamine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases. BioFactors 38:365–371. doi:10.1002/biof.1026
Nedeva I et al (2013) Synthetic polyamines promote rapid lamellipodial growth by regulating actin dynamics. Nature Commun 4:2165. doi:10.1038/ncomms3165
Nemes Z Jr, Adany R, Balazs M, Boross P, Fesus L (1997) Identification of cytoplasmic actin as an abundant glutaminyl substrate for tissue transglutaminase in HL-60 and U937 cells undergoing apoptosis. J Biol Chem 272:20577–20583
Oriol-Audit C (1978) Polyamine-induced actin polymerization. Eur J Biochem/FEBS 87:371–376
Osawa M, Yokogawa M, Muramatsu T, Kimura T, Mase Y, Shimada I (2009) Evidence for the direct interaction of spermine with the inwardly rectifying potassium channel. J Biol Chem 284:26117–26126. doi:10.1074/jbc.M109.029355
Park MH, Nishimura K, Zanelli CF, Valentini SR (2010) Functional significance of eIF5A and its hypusine modification in eukaryotes. Amino Acids 38:491–500. doi:10.1007/s00726-009-0408-7
Pegg AE (2014) The function of spermine. IUBMB Life 66:8–18. doi:10.1002/iub.1237
Peron A et al (2013) Snyder-Robinson syndrome: a novel nonsense mutation in spermine synthase and expansion of the phenotype. Am J Med Genet A 161A:2316–2320. doi:10.1002/ajmg.a.36116
Perrella FW, Lea MA (1978) Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver. Biochem Biophys Res Commun 82:575–581
Pohjanpelto P, Virtanen I, Holtta E (1981) Polyamine starvation causes disappearance of actin filaments and microtubules in polyamine-auxotrophic CHO cells. Nature 293:475–477
Ray RM, Li C, Bhattacharya S, Naren AP, Johnson LR (2012) Spermine, a molecular switch regulating EGFR, integrin beta3 Src, and FAK scaffolding. Cell Signal 24:931–942. doi:10.1016/j.cellsig.2011.12.016
Savarin P et al (2010) A central role for polyamines in microtubule assembly in cells. Biochem J 430:151–159. doi:10.1042/BJ20091811
Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, Aktories K (1997) Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387:725–729. doi:10.1038/42735
Schmidt G, Selzer J, Lerm M, Aktories K (1998) The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity. J Biol Chem 273:13669–13674
Shimizu T, Takao T, Hozumi K, Nunomura K, Ohta S, Shimonishi Y, Ikegami S (1997) Structure of a covalently cross-linked form of core histones present in the starfish sperm. Biochemistry 36:12071–12079. doi:10.1021/bi970922n
Shin DM, Kang J, Ha J, Kang HS, Park SC, Kim IG, Kim SJ (2008) Cystamine prevents ischemia-reperfusion injury by inhibiting polyamination of RhoA. Biochem Biophys Res Commun 365:509–514. doi:10.1016/j.bbrc.2007.11.007
Singh US, Kunar MT, Kao YL, Baker KM (2001) Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2. EMBO J 20:2413–2423. doi:10.1093/emboj/20.10.2413
Song Y et al (2013) Transglutaminase and polyamination of tubulin: posttranslational modification for stabilizing axonal microtubules. Neuron 78:109–123. doi:10.1016/j.neuron.2013.01.036
Sowa GZ, Cannell DS, Liu AJ, Reisler E (2006) Polyamine-induced bundling of F-actin. J Phys Chem B 110:22279–22284. doi:10.1021/jp063371w
Stamnaes J, Fleckenstein B, Sollid LM (2008) The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions. Biochim Biophys Acta 1784:1804–1811. doi:10.1016/j.bbapap.2008.08.011
Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790. doi:10.1146/annurev.bi.53.070184.003533
Tsai YH, Lai WF, Wu YW, Johnson LR (1998) Two distinct classes of rat intestinal mucosal enzymes incorporating putrescine into protein. FEBS Lett 435:251–256
Visvanathan A, Ahmed K, Even-Faitelson L, Lleres D, Bazett-Jones DP, Lamond AI (2013) Modulation of higher order chromatin conformation in mammalian cell nuclei can be mediated by polyamines and divalent cations. PloS One 8:e67689. doi:10.1371/journal.pone.0067689
Walther DJ, Stahlberg S, Vowinckel J (2011) Novel roles for biogenic monoamines: from monoamines in transglutaminase-mediated post-translational protein modification to monoaminylation deregulation diseases. FEBS J 278:4740–4755. doi:10.1111/j.1742-4658.2011.08347.x
Wang X, Ikeguchi Y, McCloskey DE, Nelson P, Pegg AE (2004) Spermine synthesis is required for normal viability, growth, and fertility in the mouse. J Biol Chem 279:51370–51375. doi:10.1074/jbc.M410471200
Wilhelm B, Meinhardt A, Seitz J (1996) Transglutaminases: purification and activity assays. J Chromatogr B Biomed Appl 684:163–177
Acknowledgments
This work was supported by grants from the National Science Council and National Taiwan University; NSC 102-2311-B-002-046-MY3, NTU-CESRP-102R7602B1, NTU-CESRP-103R7602B1, NTU-CESRP-103R8600. Proteomic mass spectrometry analyses were performed by the Core Facilities for Protein Structural Analysis located at the Institute of Biological Chemistry, Academia Sinica, supported by NSC100-2325-B-001-029 and the Academia Sinica.
Conflict of interest
The authors have no conflicts of interest to disclose.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yu, CH., Chou, CC., Lee, YJ. et al. Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis. Amino Acids 47, 469–481 (2015). https://doi.org/10.1007/s00726-014-1879-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-014-1879-8