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Specific Detection of Neu5Gc in Animal Tissues by Immunohistochemistry

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Xenotransplantation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2110))

Abstract

One of the major obstacles in xenotransplantation is tissue expression of the non-human mammalian carbohydrate N-glycolylneuraminic acid (Neu5Gc). This 9-carbon backbone acidic sugar is the hydroxylated form of N-acetylneuraminic acid (Neu5Ac), and both constitute the two most common sialic acid types in mammals. Loss of CMP-Neu5Ac hydroxylase encoding gene in humans dictates the immunogenic nature of Neu5Gc-containing xenografts. Here we describe an immunohistochemistry method for the detection of Neu5Gc in mammalian-derived tissues using affinity-purified chicken anti-Neu5Gc IgY. Specificity is further demonstrated by competitive inhibition with free Neu5Gc or Neu5Gc-glycoproteins, but not with Neu5Ac or Neu5Ac-glycoproteins. This method can be used to evaluate potential Neu5Gc-immunogenicity of xenografts.

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References

  1. Ekser B, Li P, Cooper DKC (2017) Xenotransplantation: past, present, and future. Curr Opin Organ Transplant 22:513–521

    PubMed  PubMed Central  Google Scholar 

  2. Vadori M, Cozzi E (2015) The immunological barriers to xenotransplantation. Tissue Antigens 86:239–253

    Article  CAS  PubMed  Google Scholar 

  3. Cooper DKC, Gaston R, Eckhoff D, Ladowski J, Yamamoto T, Wang L, Iwase H, Hara H, Tector M, Tector AJ (2018) Xenotransplantation-the current status and prospects. Br Med Bull 125:5–14

    Article  CAS  PubMed  Google Scholar 

  4. Byrne GW, McGregor CGA, Breimer ME (2015) Recent investigations into pig antigen and anti-pig antibody expression. Int J Surg 23:223–228

    Article  PubMed  PubMed Central  Google Scholar 

  5. Galili U (2013) Anti-Gal: an abundant human natural antibody of multiple pathogeneses and clinical benefits. Immunology 140:1–11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cooper DK (2016) Modifying the sugar icing on the transplantation cake. Glycobiology 26:571–581

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Padler-Karavani V, Varki A (2011) Potential impact of the non-human sialic acid N-glycolylneuraminic acid on transplant rejection risk. Xenotransplantation 18:1–5

    Article  PubMed  PubMed Central  Google Scholar 

  8. French BM, Sendil S, Pierson RN, Azimzadeh AM (2017) The role of sialic acids in the immune recognition of xenografts. Xenotransplantation 24

    Google Scholar 

  9. Salama A, Evanno G, Harb J, Soulillou JP (2015) Potential deleterious role of anti-Neu5Gc antibodies in xenotransplantation. Xenotransplantation 22:85–94

    Article  PubMed  Google Scholar 

  10. Reuven EM, Leviatan Ben-Arye S, Marshanski T, Breimer ME, Yu H, Fellah-Hebia I, Roussel JC, Costa C, Galiñanes M, Mañez R, Le Tourneau T, Soulillou JP, Cozzi E, Chen X, Padler-Karavani V (2016) Characterization of immunogenic Neu5Gc in bioprosthetic heart valves. Xenotransplantation 23:381–392

    Article  PubMed  PubMed Central  Google Scholar 

  11. Varki A (2010) Colloquium paper: uniquely human evolution of sialic acid genetics and biology. Proc Natl Acad Sci U S A 107(Suppl 2):8939–8946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nguyen DH, Tangvoranuntakul P, Varki A (2005) Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells. J Immunol 175:228–236

    Article  CAS  PubMed  Google Scholar 

  13. Padler-Karavani V, Yu H, Cao H, Chokhawala H, Karp F, Varki N, Chen X, Varki A (2008) Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. Glycobiology 18:818–830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gao B, Long C, Lee W, Zhang Z, Gao X, Landsittel D, Ezzelarab M, Ayares D, Huang Y, Cooper DKC, Wang Y, Hara H (2017) Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans. PLoS One 12:e0180768

    Article  PubMed  PubMed Central  Google Scholar 

  15. Okerblom J, Varki A (2017) Biochemical, Cellular, Physiological, and Pathological Consequences of Human Loss of N-Glycolylneuraminic Acid. Chembiochem 18:1155–1171

    Article  CAS  PubMed  Google Scholar 

  16. Leviatan Ben-Arye S, Yu H, Chen X, Padler-Karavani V (2017) Profiling anti-Neu5Gc IgG in human sera with a sialoglycan microarray assay. J Vis Exp 125

    Google Scholar 

  17. Paul A, Padler-Karavani V (2018) Evolution of sialic acids: implications in xenotransplant biology. Xenotransplantation 25(6):e12424

    Article  PubMed  PubMed Central  Google Scholar 

  18. Burdorf L, Azimzadeh AM, Pierson RN (2018) Progress and challenges in lung xenotransplantation: an update. Curr Opin Organ Transplant 23:621–627

    Article  CAS  PubMed  Google Scholar 

  19. Zhang R, Wang Y, Chen L, Wang R, Li C, Li X, Fang B, Ren X, Ruan M, Liu J, Xiong Q, Zhang L, Jin Y, Zhang M, Liu X, Li L, Chen Q, Pan D, Li R, Cooper DKC, Yang H, Dai Y (2018) Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH. Acta Biomater 72:196–205

    Article  CAS  PubMed  Google Scholar 

  20. Diaz SL, Padler-Karavani V, Ghaderi D, Hurtado-Ziola N, Yu H, Chen X, Brinkman-Van der Linden EC, Varki A, Varki NM (2009) Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS One 4:e4241

    Article  PubMed  PubMed Central  Google Scholar 

  21. Padler-Karavani V, Song X, Yu H, Hurtado-Ziola N, Huang S, Muthana S, Chokhawala HA, Cheng J, Verhagen A, Langereis MA, Kleene R, Schachner M, de Groot RJ, Lasanajak Y, Matsuda H, Schwab R, Chen X, Smith DF, Cummings RD, Varki A (2012) Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays. J Biol Chem 287:22593–22608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Naito-Matsui Y, Davies LR, Takematsu H, Chou HH, Tangvoranuntakul P, Carlin AF, Verhagen A, Heyser CJ, Yoo SW, Choudhury B, Paton JC, Paton AW, Varki NM, Schnaar RL, Varki A (2017) Physiological exploration of the long term evolutionary selection against expression of N-glycolylneuraminic acid in the brain. J Biol Chem 292:2557–2570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Davies LR, Varki A (2015) Why is N-glycolylneuraminic acid rare in the vertebrate brain. Top Curr Chem 366:31–54

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Springer SA, Gagneux P (2016) Glycomics: revealing the dynamic ecology and evolution of sugar molecules. J Proteome 135:90–100

    Article  CAS  Google Scholar 

  25. Peri S, Kulkarni A, Feyertag F, Berninsone PM, Alvarez-Ponce D (2018) Phylogenetic distribution of CMP-Neu5Ac hydroxylase (CMAH), the enzyme synthetizing the proinflammatory human xenoantigen Neu5Gc. Genome Biol Evol 10:207–219

    Article  CAS  PubMed  Google Scholar 

  26. Varki A (2001) Loss of N-glycolylneuraminic acid in humans: mechanisms, consequences, and implications for hominid evolution. Am J Phys Anthropol Suppl 33:54–69

    Article  Google Scholar 

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Acknowledgment

This work was supported by the 7th Framework Program FP7-Health-2013-INNOVATION-1-603049 of the European Commission (to V.P-K).

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Authors and Affiliations

  1. Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel

    Anu Paul, Shirley Bachar Abramovitch & Vered Padler-Karavani

Authors
  1. Anu Paul
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  2. Shirley Bachar Abramovitch
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  3. Vered Padler-Karavani
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Corresponding author

Correspondence to Vered Padler-Karavani .

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Editors and Affiliations

  1. Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Hospital Duran i Reynals, L’Hospitalet de LLobregat, Barcelona, Spain

    Cristina Costa

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Paul, A., Bachar Abramovitch, S., Padler-Karavani, V. (2020). Specific Detection of Neu5Gc in Animal Tissues by Immunohistochemistry. In: Costa, C. (eds) Xenotransplantation. Methods in Molecular Biology, vol 2110. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0255-3_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0255-3_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0254-6

  • Online ISBN: 978-1-0716-0255-3

  • eBook Packages: Springer Protocols

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