[Frontiers In Bioscience, Landmark, 24, 1085-1096, Jan 1, 2019]

Interactions between human hemoglobin subunits and peroxiredoxin 2

Qiang Ma1, Liang An1, Huifang Tian2, Jia Liu1, Liqiang Zhang3, Xiaojing Li1, Chunhua Wei1, Caixia Xie1, Huirong Ding2, Wenbin Qin1, Yan Su1

1Laboratory of Hemoglobin, Baotou Medical College, 014060, Baotou, China, 2Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Central Laboratory, Peking University Cancer Hospital & Institute, 100142, Beijing, China, 3State Key Laboratory of Heavy Oil Processing and Department of Materials Science and Engineering, China University of Petroleum, 102249, Beijing, China


1. Abstract
2. Introduction
3. Materials and methods
3.1. IDQD prediction method
3.2. Preparation of RBC suspension and hemolysate
3.3. Purification of different Hb components from starch-agarose mixed gel
3.4. Coimmunoprecipitation (co-IP)
3.6. In-gel digestion
3.7. Identification of proteins by liquid chromatography tandem mass spectrometry
3.8. Western blotting
3.9. XAFS experiment
4. Results
4.1. Discrimination of the interactions between human hemoglobin subunits and Prx2 based on the IDQD model
4.2. Comparison of IDQD-predicted interactions between different Hb subunits with the experimental results
4.3. Interaction between HbA and Prx2
4.4. XAFS to clarify the interaction mechanism between HbA and Prx2
5. Discussion
6. Acknowledgments
7. References


Red blood cells (RBCs) are exposed to exogenous reactive oxygen species in the circulatory system. To this end, the interactions between the different hemoglobin (Hb) subunits and peroxiredoxin 2, which is a ubiquitous member of the antioxidant enzymes that also controls the cytokine-induced peroxide levels, were assessed. We predicted by the increment of diversity with quadratic discriminant analysis (IDQD) that peroxiredoxin2 (Prx2) could interact with the hemoglobin alpha, beta and gamma subunits but not with the delta subunit. Coimmunoprecipitation (co-IP), electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry, Western blotting and X-ray absorption fine structure (XAFS) spectroscopy were performed to verify these predictions. The results showed that Prx2 was a member of the beta-globin immunoprecipitating complex that existed in hemoglobin A, hemolysate-hemoglobin A, hemoglobin A-hemoglobin A2, hemolysate-hemoglobin A-hemoglobin A2 and hemoglobin A2 but not in hemolysate-hemoglobin A2. Adding Prx2 to hemoglobin A altered the second shell of iron embedded in hemoglobin A. Therefore, Prx2 interacts with hemoglobin A (Alpha2Beta2) and hemoglobin F (Alpha2Gamma2) but not with hemoglobin A2 (Alpha2Delta2).


1. I. Sendina-Nadal, Y. Ofran, J. A. Almendral, J. M. Buldu, I. Leyva, D. Q. Li, S. Havlin and S. Boccaletti: Unveiling protein functions through the dynamics of the interaction network. PloS one 6 (3), e17679 (2011)
DOI: 10.1371/journal.pone.0017679

2. D. Bu , Y. Zhao, L. Cai , H. Xue , X. Zhu, H. Lu , J. Zhang, S. Sun, L. Ling, N. Zhang, G. Li and R. Chen: Topological structure analysis of the protein-protein interaction network in budding yeast. Nucleic Acids Res 31 (9), 2443-2450 (2003)
DOI: 10.1093/nar/gkg340

3. T. Hase, H. Tanaka, Y. Suzuki, S. Nakagawa and H. Kitano: Structure of protein interaction networks and their implications on drug design. Plos Comput Biol 5 (10), e1000550 (2009)
DOI: 10.1371/journal.pcbi.1000550

4. T. Schutze, A. K. Ulrich, L. Apelt, C. L. Will, N. Bartlick, M. Seeger, G. Weber, R. Lührmann, U.Stelzl, and M. C. Wahl: Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome. RNA (New York, NY) 22 (2), 265-277 (2015)
DOI: 10.1261/rna.054296.115

5. J. Song, J. Li, H. D. Liu, W. Liu, Y. Feng, X. T. Zhou and J. D. Li: Snapin interacts with G-protein coupled receptor PKR2. Biochemi Bioph Res Co 469, 501-506 (2016)
DOI: 10.1016/j.bbrc.2015.12.023

6. R. Hegde, S. M. Srinivasula, Z. Zhang, R. Wassell, R. Mukattash, L. Cilenti, G. DuBois, Y. Lazebnik, A. S. Zervos, T. Fernandes-Alnemri and E. S. Alnemri: Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 277 (1), 432-438. (2002).
DOI: 10.1074/jbc.M109721200

7. G. H. Liu , H. B. Shen and D. J. Yu: Prediction of protein-protein interaction sites with machine-learning-based data-cleaning and post-filtering procedures. J Membr Biol 249, 141-153 (2016)
DOI: 10.1007/s00232-015-9856-z

8. P. Fariselli, F. Pazos, A. Valencia and R. Casadio: Prediction of protein-protein interaction sites in heterocomplexes with neural networks. European journal of biochemistry / FEBS 269 (5), 1356-1361 (2002)
DOI: 10.1046/j.1432-1033.2002.02767.x

9. B. Wang, P. Chen, D. S. Huang, J. J. Li, T. M. Lok and M. R. Lyu: Predicting protein interaction sites from residue spatial sequence profile and evolution rate. FEBS letters 580 (2), 380-384 (2006)
DOI: 10.1016/j.febslet.2005.11.081

10. J. Jia, X. Xiao and B. Liu: Prediction of Protein-Protein Interactions with Physicochemical Descriptors and Wavelet Transform via Random Forests. J Lab Autom 21 (3), 368-377 (2016)
DOI: 10.1177/2211068215581487

11. S. H. Park, J. A. Reyes, D. R. Gilbert, J. W. Kim and S. Kim: Prediction of protein-protein interaction types using association rule based classification. BMC bioinformatics 10, 36 (2009)
DOI: 10.1186/1471-2105-10-36

12. E. Sprinzak and H. Margalit: Correlated sequence-signatures as markers of protein-protein interaction. J Mol Biol 11 (4), 681-692 (2001).
DOI: 10.1006/jmbi.2001.4920

13. L. Zhang and L. Luo: Splice site prediction with quadratic discriminant analysis using diversity measure. Nucleic Acids Res 31 (21), 6214-6220 (2003)
DOI: 10.1093/nar/gkg805

14. J. Lu , L. Luo: Prediction for human transcription start site using diversity measure with quadratic discriminant. Bioinformation 2 (7), 316-321 (2008)
DOI: 10.6026/97320630002316

15. W. Chen, Luo, L. Zhang and H. Lin: Recognition of DNase I hypersensitive sites in multiple cell lines. Int J Logist-Res App 5 (4), 378-384 (2009)
DOI: 10.1504/IJBRA.2009.027508

16. Y. Feng, L. Luo: Use of tetrapeptide signals for protein secondary-structure prediction. Amino Acids 35 (3), 607-614 (2008)
DOI: 10.1007/s00726-008-0089-7

17. W. Chen, L. Luo: Classification of antimicrobial peptide using diversity measure with quadratic discriminant analysis. J Microbiol Methods 78 (1), 94-96 (2009)
DOI: 10.1016/j.mimet.2009.03.013

18. W. Chen, L. Luo and L. Zhang: The organization of nucleosomes around splice sites. Nucleic Acids Res 38 (9), 2788-2798 (2010)
DOI: 10.1093/nar/gkq007

19. Y. Xing, X. Zhao and L. Cai: Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function. Genomics 98 (5), 359-366 (2011)
DOI: 10.1016/j.ygeno.2011.07.008

20. X. Zhao, Z. Pei, J. Liu, S.Qin and L. Cai: Prediction of nucleosome DNA formation potential and nucleosome positioning using increment of diversity combined with quadratic discriminant analysis. Chromosome Res 18 (7), 777-85 (2010)
DOI: 10.1007/s10577-010-9160-9

21. G. Liu, J. Liu, X. Cui and L. Cai: Sequence-dependent prediction of recombination hotspots in Saccharomyces cerevisiae. J theoretical biol 293, 49-54 (2012)
DOI: 10.1016/j.jtbi.2011.10.004

22. Y. Su, G. Shao, L. Gao, L. Zhou, L. Qin and W. Qin: RBC electrophoresis with discontinuous power supply a newly established hemoglobin release test. Electrophoresis 30 (17), 3041-43 (2009)
DOI: 10.1002/elps.200900176

23. T. H. Lee, S. U. Kim, S. L.Yu, S. H. Kim, D. S. Park, H. B. Moon, S. H. Dho, K. S. Kwon, H. J. Kwon, Y. H. Han, S. Jeong, S. W. Kang , H. S. Shin , K. K. Lee , S. G. Rhee and D. Y. Yu: Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice. Blood 101 (12), 5033-38 (2003)
DOI: 10.1182/blood-2002-08-2548

24. D. G. Kakhniashvili, Jr. Bulla L. A. and S. R. Goodman: The human erythrocyte proteome: analysis by ion trap mass spectrometry. Mol Cell Proteomics 3 (5), 501-509 (2004)
DOI: 10.1074/mcp.M300132-MCP200

25. R. M. Johnson, Jr. Goyette G., Y. Ravindranath and Y. S. Ho: Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes. Free Radic Biol Med 39 (11), 1407-1417 (2005)
DOI: 10.1016/j.freeradbiomed.2005.07.002

26. K. M. Stuhlmeier, J. J. Kao, P. Wallbrandt, M. Lindberg, B. Hammarstrom , H. Broell and B. Paigen: Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates. Eur J Biochem / FEBS 270 (2), 334-341 (2003)
DOI: 10.1046/j.1432-1033.2003.03393.x

27. F. M. Low , M. B. Hampton and C. C. Winterbourn: Peroxiredoxin 2 and peroxide metabolism in the erythrocyte. Antioxid Redox Signal 10 (9), 1621-1630 (2008)
DOI: 10.1089/ars.2008.2081

28. M. Brizuela, H. M. Huang, C. Smith, G. Burgio, S. J. Foote and B. J. McMorran: Treatment of erythrocytes with the 2-cys peroxiredoxin inhibitor, Conoidin A, prevents the growth of Plasmodium falciparum and enhances parasite sensitivity to chloroquine. PloS one 9 (4), e92411 (2014)
DOI: 10.1371/journal.pone.0092411

29. S. B. Bayer , G. Maghzal , R. Stocker , M. B. Hampton and C. C. Winterbourn: Neutrophil-mediated oxidation of erythrocyte peroxiredoxin 2 as a potential marker of oxidative stress in inflammation. FASEB J 27 (8), 3315-3322 (2013)
DOI: 10.1096/fj.13-227298

30. Y. Su, L. Gao, Q. Ma, L. Zhou, L. Qin, L. Han and W. B. Qin: Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test. Electrophoresis 31 (17), 2913-20 (2010)
DOI: 10.1002/elps.201000034

31. A. Basu and A. Chakrabarti: Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction. J Proteomics 128: 469-75 (2015)
DOI: 10.1016/j.jprot.2015.06.014

32. L. Salwinski, C. S. Miller, A. J. Smith, F. K. Pettit, J. U. Bowie and D. Eisenberg: The Database of Interacting Proteins: 2004 update. Nucleic Acids Res 32 (Database issue), D449-451 (2004)
DOI: 10.1093/nar/gkh086

33. B. E. Suzek, H. Huang, P. McGarvey, R. Mazumder and C. H. WuUniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics 23 (10), 1282-1288 (2007)
DOI: 10.1093/bioinformatics/btm098

34. L. Cai, J. Liu and X. Zhao: Protein-Protein Interaction Prediction Using Increment of Diversity Combined with Quadratic Discriminant Analysis. The 2010 International Congress on Computer Applications and Computational Science; Singapore; 613-616 (2010)

35. Y. Su, L. Gao and W. Qin: Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test. Methods Mol Biol 869, 393-402 (2012)
DOI: 10.1007/978-1-61779-821-4_32

36. Y. Su, J. Shen, L. Gao, H. F. Tian, Z. H Tian and W. B. Qin: Molecular interactions of re-released proteins in electrophoresis of human erythrocytes. Electrophoresis, 33 (9-10),1402-05 (2012)
DOI: 10.1002/elps.201100644

37. J. M. Tranquada and R. Ingalls: Extended x-ray\char22{}absorption fine-structure study of anharmonicity in CuBr. Physical Review B, 28 (6), 3520-28 (1983)
DOI: 10.1103/PhysRevB.28.3520

38. L. R. Manning, J. E. Russell, J. C. Padovan, B. T. Chait, A. Popowicz, R. S. Manning and J. M. Manning: Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell. Protein Sci 16 (8), 1641-1658 (2007)
DOI: 10.1110/ps.072891007

39. L. R. Manning, J. E. Russell, A. M. Popowicz, R. S. Manning, J. C. Padovan and J. M. Manning: Energetic differences at the subunit interfaces of normal human hemoglobins correlate with their developmental profile. Biochemistry, 48 (32), 7568-74 (2009)
DOI: 10.1021/bi900857r

40. MJ. McDonald: Assembly of human adult and sickle hemoglobins from their oxygenated subunits. Differential rates of beta chain tetramer dissociation. J Biol Chem 256 (12), 6487-6490 (1981)

41. E. R. Huehns and E. M. Shooter :Human Haemoglobins. J Med Genet 2 (1), 48-90 (1965)
DOI: 10.1136/jmg.2.1.48

42. E. R. Huehns, G. H. Beaven and B. L. Stevens: Reaction of haemoglobin alpha-A with haemoglobins beta-A4, gamma-F4 and delta-A2. Biochem J 92 (2):444-448 (1964)
DOI: 10.1042/bj0920444

43. R. M. Nalbandian, R. L. Henry, L. F. J. R. Camp, P. L. Wolf and T. N. Evans: Embryonic, fetal, and neonatal hemoglobin synthesis: relationship to abortion and thalassemia. Obstet Gynecol Surv 26 (2), 184-191 (1971)
DOI: 10.1097/00006254-197102000-00027

44. U. Sen, J. Dasgupta, D. Choudhury, P. Datta, A. Chakrabarti, S. B. Chakrabarty, A. Chakrabarty and J. K Dattagupta: Crystal structures of HbA2 and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of HbA2 and identification of the ferrocyanide binding site in Hb. Biochemistry, 43 (39), 12477-12488 (2004)
DOI: 10.1021/bi048903i

45. L. R. Manning, A. M. Popowicz, J. Padovan, B. T. Chait, J. E. Russell and J. M. Manning: Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces. Protein Sci 19 (8), 1595-1599 (2010)
DOI: 10.1002/pro.441

46. E. P. Vichinsky: Clinical manifestations of alpha-thalassemia. Cold Spring Harb Perspect Med 3 (5),a011742 (2013)
DOI: 10.1101/cshperspect.a011742

47. D. R. Higgs: The molecular basis of alpha-thalassemia. Cold Spring Harb Perspect Med 3 (1), a011718 (2013)
DOI: 10.1101/cshperspect.a011718

48. C. Jin, Y. Li, Y. L. Li, Y. Zou, G. L. Zhang, M. Normura and G. Y. Zhu: Blood lead: Its effect on trace element levels and iron structure in hemoglobin. Nucl Instrum Meth B 266 (16), 3607-3613 (2008)
DOI: 10.1016/j.nimb.2008.05.087

49. P. D’Angelo and M. Benfatto: Effect of Multielectronic ConFigurations on the XAFS Analysis at the Fe K Edge. J Phys Chem A 108 (20), 4505-4514 (2004)
DOI: 10.1021/jp0499732

50. P. Eisenberger, R. G. Shulman, B. M. Kincaid, G. S. Brown and S. Ogawa: Extended X-ray absorption fine structure determination of iron nitrogen distances in haemoglobin. Nature, 274 (5666):30-34 (1978)
DOI: 10.1038/274030a0

51. L.Laraia, G. McKenzie, D. R. Spring, A. R. Venkitaraman and D. J. Huggins: Overcoming Chemical, Biological, and Computational Challenges in the Development of Inhibitors Targeting Protein-Protein Interactions, Chem Biol 22 (6), 689-703 (2015)
DOI: 10.1016/j.chembiol.2015.04.019

52. S. Martin, D. Roe and J. L. Faulon: Predicting protein-protein interactions using signature products. Bioinformatics 21 (2), 218-226 (2005)
DOI: 10.1093/bioinformatics/bth483

53. E. Schroder, J. A. Littlechild, A. A. Lebedev, N. Errington, A. A. Vagin and M. N: Isupov Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution. Structure 8 (6), 605-15 (2000)
DOI: 10.1016/S0969-2126(00)00147-7

54. T. L. Sorensen, K. E. McAuley, R. Flaig and E. M. Duke: New light for science synchrotron radiation in structural medicine. Trends in biotechnology 24 (11), 500-08 (2006)
DOI: 10.1016/j.tibtech.2006.09.006

55. Z.Wu, R. E. Benfield, Y. Wang, L. Guo, M.Tan, H. Zhang, Y. Ge and D. Grandiean: EXAFS study on the local atomic structures around iron in glycosylated haemoglobin. Phys Med Biol 46 (3), N71-77 (2001)
DOI: 10.1088/0031-9155/46/3/403

56. E. Nagababu, J. G. Mohanty, J. S. Friedman and J. M. Rifkind: Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress. Free Radic Res 47 (3), 164-71 (2013).
DOI: 10.3109/10715762.2012.756138

57. A. Matte, M. Bertoldi, N. Mohandas, X. L. An, A. Bugatti, A. M. Brunati, M. Rusnati, E. Tibaldi, A. Siciliano, F. Turrini, S. Perrotta and L. D. Franceschi: Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3. Free Radic Biol Med 55, 27-35 (2013).
DOI: 10.1016/j.freeradbiomed.2012.10.543

58. Y. H. Han, S. U. Kim, T. H. Kwon, D. S. Lee, H. L. Ha, D. S. Park, E. J. Woo, S. H. Lee, J. M. Kim, H. B. Chae, S. Y.Lee, B. Y. Kim,D. Y. Yoon, S. G. Rhee, E.Fibach and D. Y. Yu: Peroxiredoxin II is essential for preventing hemolytic anemia from oxidative stress through maintaining hemoglobin stability. Biochem Biophys Res Commun 426 (3), 427-32 (2012).
DOI: 10.1016/j.bbrc.2012.08.113

59. F. C. Cheah, A. V. Peskin, F. L. Wong, A. Ithnin, A. Othman and C. C. Winterbourn: Increased basal oxidation of peroxiredoxin 2 and limited peroxiredoxin recycling in glucose-6-phosphate dehydrogenase-deficient erythrocytes from newborn infants. FASEB J, 28 (7): 3205-10 (2014).
DOI: 10.1096/fj.14-250050

60. A. Mattea, P. S. Lowb, F. Turrinic, M. Bertoldid, M. E. Campanellab, D. Spanoe, A. Pantaleoa,f, A.Sicilianoa and L. D. Franceschia: Peroxiredoxin-2 expression is increased in beta-thalassemic mouse red cells but is displaced from the membrane as a marker of oxidative stress. Free Radic Biol Med, 49 (3): 457-66 (2010).
DOI: 10.1016/j.freeradbiomed.2010.05.003

61. S. Rocha, R. M. P. Vitorino, F. M. Lemos-Amado, E. B. Castro, P. Rocha-Pereira, J. Barbot, E. Cleto, F. Ferreira, A. Quintanilha, L. Belo, A. Santos-Silva : Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis. Blood Cells Mol Dis, 41 (1): 5-9 (2008).
DOI: 10.1016/j.bcmd.2008.02.008

62. S. Rinalducci, G. M. D’Amici, B. Blasi, S. Vaglio, G. Grazzini and L. Zolla: Peroxiredoxin-2 as a candidate biomarker to test oxidative stress levels of stored red blood cells under blood bank conditions. Transfusion, 51 (7): 1439-49 (2011).
DOI: 10.1111/j.1537-2995.2010.03032.x

63. J. Y. Oh, V. Harper, C. W. Sun, R. Stapley, L. Wilson, M. B. Marques, S. Barnes, T. Townes and R. P. PaTel: Peroxiredoxin-2 recycling is inhibited during erythrocyte storage. Antioxid Redox Signal, 22 (4): 294-307 (2015).
DOI: 10.1089/ars.2014.5950

Abbreviations: Reactive oxygen species (ROS), hemoglobin (Hb), peroxiredoxin2 (Prx2), increment of diversity with quadratic discriminant analysis (IDQD), co-immunoprecipitation (co-IP), electrospray ionization quadrupole-time of flight (ESI-Q-TOF), X-ray absorption fine structure (XAFS), protein-protein interactions (PPIs), support vector machines (SVMs), random forests (RFs), red blood cells (RBCs), reactive oxygen species (ROSs), glutathione peroxidase (GSH-Px), peroxiredoxins (Prxs), X-ray absorption near-edge structure (XANES), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), trifluoroacetic acid (TFA).

Key Words: Peroxiredoxin 2, Hemoglobin, Interaction, Increment Of Diversity With Quadratic Discriminant Analysis, X-Ray Absorption Fine Structure

Send correspondence to: Yan Su, Laboratory of Hemoglobin, Baotou Medical College, 014060, Baotou, Inner Mongolia, China, Tel: 86-472-7167834, Fax: 86-472-7167834, E-mail: synmg@126.com