Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Differential Sialic Acid Content and Hemoglobin-binding Activity of Precursor Prohaptoglobin and Mature Haptoglobin

전구체 프로합토글로빈과 성숙 합토글로빈의 시알산 함량 및 헤모글로빈-결합력 비교

  • Lee, Joo-Hyun (Department of Medical Lifescience, College of Medicine, The Catholic University of Korea) ;
  • Oh, Mi-Kyung (Department of Medical Lifescience, College of Medicine, The Catholic University of Korea) ;
  • Kim, In-Sook (Department of Medical Lifescience, College of Medicine, The Catholic University of Korea)
  • 이주현 (가톨릭대학교 의과대학 의생명과학교실) ;
  • 오미경 (가톨릭대학교 의과대학 의생명과학교실) ;
  • 김인숙 (가톨릭대학교 의과대학 의생명과학교실)
  • Received : 2017.04.07
  • Accepted : 2017.05.11
  • Published : 2017.06.30
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Abstract

Mature haptoglobin (Hp) is a plasma glycoprotein and acts as an antioxidant by scavenging cell-free hemoglobin (Hb). Prohaptoglobin (proHp) is an unprocessed Hp precursor which is present a little in circulation. However, the biological function of proHp remains unknown. To investigate the structural and functional differences between proHp and Hp, we prepared recombinant proHp isoforms and compared their sialic acid content and Hb-binding capacity with those of mature isoforms. When proHp samples were analyzed by Western blot under non-reducing conditions, proHp1 was detected as one band of approximately 130 kDa and proHp2 as multiple bands >200 kDa, in the manner of mature Hp1-1 and Hp2-2, respectively. On the native polyacrylamide gel under non-reducing and non-denaturing conditions, both proHp isoforms migrated more slowly than their mature Hp counterparts. In addition, the lectin-based ELISA assay demonstrated that the content of sialic acid in proHp1 and proHp2 was much less than in Hp1-1 and Hp2-2. The Hb-binding capacity of proHp was also lower than those of mature Hp. These findings indicate that proHp and Hp are similar in the size and polymerization pattern, but different in sialic acid content and Hb-binding activity. It suggests precursor proHp may exert different functions in circulation than does mature Hp.

성숙 합토글로빈(haptoglobin, Hp)은 혈장 당단백질로서 혈중의 유리 헤모글로빈(hemoglobin, Hb)을 제거하고 항산화 작용을 한다. 프로합토글로빈(prohaptoglobin, proHp)은 Hp 전구체이며 혈중에 낮은 농도로 존재하지만 그 생리적 기능은 아직 확실하지 않다. 본 연구에서는 proHp와 Hp의 구조적, 기능적 차이를 연구하기 위하여 재조합 proHp를 제조하여 시알산과 Hb-결합력을 조사하고 성숙 Hp와 비교하였다. 비환원 조건의 Western blot 분석에서 proHp1은 약 130 kD의 하나의 밴드를 보였고 proHp2는 200 kDa 이상의 다중 밴드를 보였는데 이것은 대응되는 성숙 Hp1-1과 Hp2-2와 각각 동일한 양상이었다. 하지만 비환원 및 비변성 조건 하에서 수행된 전기영동에서는 proHp가 Hp보다 더 느리게 이동하였다. 렉틴을 이용한 ELISA 분석에서 proHp는 Hp보다 산성 당인 시 알산 함량이 더 낮음을 알 수 있었다. 또한 proHp는 Hb과의 결합력도 더 낮았다. 이러한 결과들로부터 proHp는 Hp와 같은 양상으로 중합체를 형성하지만 시알산 함량과 Hb-결합력이 Hp와 다름을 알 수 있었고 혈중에서 전구체인 proHp는 Hp와 다른 기능을 수행할 것임을 시사한다.

Keywords

References

  1. Andersen, C. B., Torvund-Jensen, M., Nielsen, M. J., de Oliveira, C. L., Hersleth, H. P., Andersen, N. H., Pedersen, J. S., Andersen, G. R. and Moestrup, S. K. 2012. Structure of the haptoglobin-haemoglobin complex. Nature 489, 456-459. https://doi.org/10.1038/nature11369
  2. Ang, I. L., Poon, T. C., Lai, P. B., Chan, A. T., Ngai, S. M., Hui, A. Y., Johnson, P. J. and Sung, J. J. 2006. Study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach. J. Proteome Res. 5, 2691-2700. https://doi.org/10.1021/pr060109r
  3. Arredouani, M., Matthijs, P., Van Hoeyveld, E., Kasran, A., Baumann, H., Ceuppens, J. L. and Stevens, E. 2003. Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release. Immunology 108, 144-151. https://doi.org/10.1046/j.1365-2567.2003.01569.x
  4. Arredouani, M. S., Kasran, A., Vanoirbeek, J. A., Berger, F. G., Baumann, H. and Ceuppens, J. L. 2005. Haptoglobin dampens endotoxin-induced inflammatory effects both in vitro and in vivo. Immunology 114, 263-271. https://doi.org/10.1111/j.1365-2567.2004.02071.x
  5. Bowman, B. H. and Kurosky, A. 1982. Haptoglobin: the evolutionary product of duplication, unequal crossing over, and point mutation. Adv. Hum. Genet. 12, 189-261, 453-184.
  6. Cid, M. C., Grant, D. S., Hoffman, G. S., Auerbach, R., Fauci, A. S. and Kleinman, H. K. 1993. Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J. Clin. Invest. 91, 977-985. https://doi.org/10.1172/JCI116319
  7. Galicia, G., Maes, W., Verbinnen, B., Kasran, A., Bullens, D., Arredouani, M. and Ceuppens, J. L. 2009. Haptoglobin deficiency facilitates the development of autoimmune inflammation. Eur. J. Immunol. 39, 3404-3412. https://doi.org/10.1002/eji.200939291
  8. Hanley, J. M., Haugen, T. H. and Heath, E. C. 1983. Biosynthesis and processing of rat haptoglobin. J. Biol. Chem. 258, 7858-7869.
  9. Huntoon, K. M., Russell, L., Tracy, E., Barbour, K. W., Li, Q., Shrikant, P. A., Berger, F. G., Garrett-Sinha, L. A. and Baumann, H. 2013. A unique form of haptoglobin produced by murine hematopoietic cells supports B-cell survival, differentiation and immune response. Mol. Immunol. 55, 345-354. https://doi.org/10.1016/j.molimm.2013.03.008
  10. Kormoczi, G. F., Saemann, M. D., Buchta, C., Peck- Radosavljevic, M., Mayr, W. R., Schwartz, D. W., Dunkler, D., Spitzauer, S. and Panzer, S. 2006. Influence of clinical factors on the haemolysis marker haptoglobin. Eur. J. Clin. Invest. 36, 202-209. https://doi.org/10.1111/j.1365-2362.2006.01617.x
  11. Kristiansen, M., Graversen, J. H., Jacobsen, C., Sonne, O., Hoffman, H. J., Law, S. K. and Moestrup, S. K. 2001. Identification of the haemoglobin scavenger receptor. Nature 409, 198-201. https://doi.org/10.1038/35051594
  12. Langlois, M. R. and Delanghe, J. R. 1996. Biological and clinical significance of haptoglobin polymorphism in humans. Clin. Chem. 42, 1589-1600.
  13. Levy, A. P., Asleh, R., Blum, S., Levy, N. S., Miller-Lotan, R., Kalet-Litman, S., Anbinder, Y., Lache, O., Nakhoul, F. M., Asaf, R., Farbstein, D., Pollak, M., Soloveichik, Y. Z., Strauss, M., Alshiek, J., Livshits, A., Schwartz, A., Awad, H., Jad, K. and Goldenstein, H. 2010. Haptoglobin: basic and clinical aspects. Antioxid. Redox Signal. 12, 293-304. https://doi.org/10.1089/ars.2009.2793
  14. Nagel, R. L. and Gibson, Q. H. 1971. The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin. J. Biol. Chem. 246, 69-73.
  15. Oh, M. K., Park, H. J., Lee, J. H., Bae, H. M. and Kim, I. S. 2015. Single chain precursor prohaptoglobin promotes angiogenesis by upregulating expression of vascular endothelial growth factor (VEGF) and VEGF receptor2. FEBS Lett. 589, 1009-1017. https://doi.org/10.1016/j.febslet.2015.03.006
  16. Okuyama, N., Ide, Y., Nakano, M., Nakagawa, T., Yamanaka, K., Moriwaki, K., Murata, K., Ohigashi, H., Yokoyama, S., Eguchi, H., Ishikawa, O., Ito, T., Kato, M., Kasahara, A., Kawano, S., Gu, J., Taniguchi, N. and Miyoshi, E. 2006. Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation. Int. J. Cancer 118, 2803-2808. https://doi.org/10.1002/ijc.21728
  17. Park, H. J., Oh, M. K., Kim, N. H., Cho, M. L. and Kim, I. S. 2013. Identification of a specific haptoglobin C-terminal fragment in arthritic synovial fluid and its effect on interleukin- 6 expression. Immunology 140, 133-141. https://doi.org/10.1111/imm.12125
  18. Philippidis, P., Mason, J. C., Evans, B. J., Nadra, I., Taylor, K. M., Haskard, D. O. and Landis, R. C. 2004. Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte- macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circ. Res. 94, 119-126. https://doi.org/10.1161/01.RES.0000109414.78907.F9
  19. Quimby, K. R., Hambleton, I. R. and Landis, R. C. 2015. Intravenous infusion of haptoglobin for the prevention of adverse clinical outcome in Sickle Cell Disease. Med. Hypotheses 85, 424-432. https://doi.org/10.1016/j.mehy.2015.06.023
  20. Ratanasopa, K., Chakane, S., Ilyas, M., Nantasenamat, C. and Bulow, L. 2013. Trapping of human hemoglobin by haptoglobin: molecular mechanisms and clinical applications. Antioxid. Redox Signal. 18, 2364-2374. https://doi.org/10.1089/ars.2012.4878
  21. Schaer, C. A., Schoedon, G., Imhof, A., Kurrer, M. O. and Schaer, D. J. 2006. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the non-inflammatory and protective transcriptional response of macrophages to hemoglobin. Circ. Res. 99, 943-950. https://doi.org/10.1161/01.RES.0000247067.34173.1b
  22. Schaer, D. J., Buehler, P. W., Alayash, A. I., Belcher, J. D. and Vercellotti, G. M. 2013. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 121, 1276-1284. https://doi.org/10.1182/blood-2012-11-451229
  23. Shen, H., Heuzey, E., Mori, D. N., Wong, C. K., Colangelo, C. M., Chung, L. M., Bruce, C., Slizovskiy, I. B., Booth, C. J., Kreisel, D. and Goldstein, D. R. 2015. Haptoglobin enhances cardiac transplant rejection. Circ. Res. 116, 1670-1679. https://doi.org/10.1161/CIRCRESAHA.116.305406
  24. Shen, H., Song, Y., Colangelo, C. M., Wu, T., Bruce, C., Scabia, G., Galan, A., Maffei, M. and Goldstein, D. R. 2012. Haptoglobin activates innate immunity to enhance acute transplant rejection in mice. J. Clin. Invest. 122, 383-387. https://doi.org/10.1172/JCI58344
  25. Tripathi, A., Lammers, K. M., Goldblum, S., Shea-Donohue, T., Netzel-Arnett, S., Buzza, M. S., Antalis, T. M., Vogel, S. N., Zhao, A., Yang, S., Arrietta, M. C., Meddings, J. B. and Fasano, A. 2009. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc. Natl. Acad. Sci. USA 106, 16799-16804. https://doi.org/10.1073/pnas.0906773106
  26. Wang, Y., Kinzie, E., Berger, F. G., Lim, S. K. and Baumann, H. 2001. Haptoglobin, an inflammation-inducible plasma protein. Redox Rep. 6, 379-385. https://doi.org/10.1179/135100001101536580
  27. Wicher, K. B. and Fries, E. 2004. Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein. Proc. Natl. Acad. Sci. USA 101, 14390-14395. https://doi.org/10.1073/pnas.0405692101
  28. Wu, J., Zhu, J., Yin, H., Buckanovich, R. J. and Lubman, D. M. 2014. Analysis of glycan variation on glycoproteins from serum by the reverse lectin-based ELISA assay. J. Proteome Res. 13, 2197-2204. https://doi.org/10.1021/pr401061c
  29. Zhang, S., Shang, S., Li, W., Qin, X. and Liu, Y. 2016. Insights on N-glycosylation of human haptoglobin and its association with cancers. Glycobiology 26, 684-692. https://doi.org/10.1093/glycob/cww016
  30. Zhao, X., Song, S., Sun, G., Strong, R., Zhang, J., Grotta, J. C. and Aronowski, J. 2009. Neuroprotective role of haptoglobin after intracerebral hemorrhage. J. Neurosci. 29, 15819-15827. https://doi.org/10.1523/JNEUROSCI.3776-09.2009