Comparative N-Linked Glycan Analysis of Wild-Type and α1,3-Galactosyltransferase Gene Knock-Out Pig Fibroblasts Using Mass Spectrometry Approaches

  • Park, Hae-Min (School of Chemical and Biological Engineering, Seoul National University) ;
  • Kim, Yoon-Woo (Department of Chemical Engineering, Soongsil University) ;
  • Kim, Kyoung-Jin (Department of Chemical Engineering, Soongsil University) ;
  • Kim, Young June (Department of Nanobiomedical Science and BK21+ NBM Global Research Center for Regenerative Medicine, Dankook University) ;
  • Yang, Yung-Hun (Department of Microbial Engineering, College of Engineering, Konkuk University) ;
  • Jin, Jang Mi (Division of Mass Spectrometry Research, Korea Basic Science Institute) ;
  • Kim, Young Hwan (Division of Mass Spectrometry Research, Korea Basic Science Institute) ;
  • Kim, Byung-Gee (School of Chemical and Biological Engineering, Seoul National University) ;
  • Shim, Hosup (Department of Nanobiomedical Science and BK21+ NBM Global Research Center for Regenerative Medicine, Dankook University) ;
  • Kim, Yun-Gon (Department of Chemical Engineering, Soongsil University)
  • Received : 2014.09.02
  • Accepted : 2014.10.21
  • Published : 2015.01.31


Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after ${\alpha}1,3$-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (${\alpha}$-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.


GalT-KO pig fibroblast;mass spectrometry (MS);N-glycan;N-glycolylneuraminic acid (NeuGc)


Supported by : National Research Foundation of Korea (NRF)


  1. Ahn, Y., Kang, U.B., Kim, J., and Lee, C. (2010). Mining of serum glycoproteins by an indirect approach using cell line secretome. Mol. Cells 29, 123-130.
  2. Ahn, K.S., Kim, Y.J., Kim, M., Lee, B.H., Heo, S.Y., Kang, M.J., Kang, Y.K., Lee, J.W., Lee, K.K., Kim, J.H., et al. (2011). Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts. Theriogenology 75, 933-939.
  3. Baumann, B.C., Stussi, G., Huggel, K., Rieben, R., and Seebach, J.D. (2007). Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs. Transplantation 83, 193-201.
  4. Burlak, C., Bern, M., Brito, A.E., Isailovic, D., Wang, Z.Y., Estrada, J.L., Li, P., and Tector, A.J. (2013). N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens. Xenotransplantation 20, 277-291.
  5. Crocker, P.R., and Feizi, T. (1996). Carbohydrate recognition systems: functional triads in cell-cell interactions. Curr. Opin. Struct. Biol. 6, 679-691.
  6. Dai, Y., Vaught, T.D., Boone, J., Chen, S.H., Phelps, C.J., Ball, S., Monahan, J.A., Jobst, P.M., McCreath, K.J., Lamborn, A.E., et al. (2002). Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat. Biotechnol. 20, 251-255.
  7. Diswall, M., Angstrom, J., Karlsson, H., Phelps, C.J., Ayares, D., Teneberg, S., and Breimer, M.E. (2010). Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies. Xenotransplantation 17, 48-60.
  8. Dube, D.H., and Bertozzi, C.R. (2005). Glycans in cancer and inflammation-- potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4, 477-488.
  9. Fujimura, T., Takahagi, Y., Shigehisa, T., Nagashima, H., Miyagawa, S., Shirakura, R., and Murakami, H. (2008). Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen- deficient cells. Mol. Rep. Dev. 75, 1372-1378.
  10. Gil, G.C., Kim, Y.G., and Kim, B.G. (2008). A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 379, 45-59.
  11. Gil, G.C., Iliff, B., Cerny, R., Velander, W.H., and Van Cott, K.E. (2010). High throughput quantification of N-glycans using onepot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Anal. Chem. 82, 6613-6620.
  12. Haslam, S.M., North, S.J., and Dell, A. (2006). Mass spectrometric analysis of N- and O-glycosylation of tissues and cells. Curr. Opin. Struct. Biol. 16, 584-591.
  13. Jang, K.S., Kim, Y.G., Gil, G.C., Park, S.H., and Kim, B.G. (2009). Mass spectrometric quantification of neutral and sialylated Nglycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines. Anal. Biochem. 386, 228-236.
  14. Kang, P., Mechref, Y., Klouckova, I., and Novotny, M.V. (2005). Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun. Mass Spectrom. 19, 3421-3428.
  15. Kang, P., Mechref, Y., and Novotny, M.V. (2008). High-throughput solid-phase permethylation of glycans prior to mass spectrometry. Rapid Commun. Mass Spectrom. 22, 721-734.
  16. Kim, Y.G., Kim, S.Y., Hur, Y.M., Joo, H.S., Chung, J., Lee, D.S., Royle, L., Rudd, P.M., Dwek, R.A., Harvey, D.J., et al. (2006). The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney. Proteomics 6, 1133-1142.
  17. Kim, Y.G., Gil, G.C., Harvey, D.J., and Kim, B.G. (2008). Structural analysis of alpha-Gal and new non-Gal carbohydrate epitopes from specific pathogen-free miniature pig kidney. Proteomics 8, 2596-2610.
  18. Kim, Y.G., Gil, G.C., Jang, K.S., Lee, S., Kim, H.I., Kim, J.S., Chung, J., Park, C.G., Harvey, D.J., and Kim, B.G. (2009a). Qualitative and quantitative comparison of N-glycans between pig endothelial and islet cells by high-performance liquid chromatography and mass spectrometry-based strategy. J. Mass Spectrom. 44, 1087-1104.
  19. Kim, Y.G., Oh, J.Y., Gil, G.C., Kim, M.K., Ko, J.H., Lee, S., Lee, H.J., Wee, W.R., and Kim, B.G. (2009b). Identification of alpha-Gal and non-Gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry. Curr. Eye Res. 34, 877-895.
  20. Kolber-Simonds, D., Lai, L., Watt, S.R., Denaro, M., Arn, S., Augenstein, M.L., Betthauser, J., Carter, D.B., Greenstein, J.L., Hao, Y., et al. (2004). Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc. Natl. Acad. Sci. USA 101, 7335-7340.
  21. Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., et al. (2002). Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089-1092.
  22. Lutz, A.J., Li, P., Estrada, J.L., Sidner, R.A., Chihara, R.K., Downey, S.M., Burlak, C., Wang, Z.Y., Reyes, L.M., Ivary, B., et al. (2013). Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation 20, 27-35.
  23. Marth, J.D., and Grewal, P.K. (2008). Mammalian glycosylation in immunity. Nat. Rev. Immunol. 8, 874-887.
  24. Miyagawa, S., Takeishi, S., Yamamoto, A., Ikeda, K., Matsunari, H., Yamada, M., Okabe, M., Miyoshi, E., Fukuzawa, M., and Nagashima, H. (2010). Survey of glycoantigens in cells from alpha1- 3galactosyltransferase knockout pig using a lectin microarray. Xenotransplantation 17, 61-70.
  25. Miyagawa, S., Maeda, A., Takeishi, S., Ueno, T., Usui, N., Matsumoto, S., Okitsu, T., Goto, M., and Nagashima, H.A (2013). lectin array analysis for wild-type and alpha-Gal-knockout pig islets versus healthy human islets. Surg. Today 43, 1439-1447.
  26. Morozumi, K., Kobayashi, T., Usami, T., Oikawa, T., Ohtsuka, Y., Kato, M., Takeuchi, O., Koyama, K., Matsuda, H., Yokoyama, I., et al. (1999). Significance of histochemical expression of Hanganutziu-Deicher antigens in pig, baboon and human tissues. Transplant Proc. 31, 942-944.
  27. North, S.J., Hitchen, P.G., Haslam, S.M., and Dell, A. (2009). Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr. Opin. Struct. Biol. 19, 498-506.
  28. Ohtsubo, K., and Marth, J.D. (2006). Glycosylation in cellular mechanisms of health and disease. Cell 126, 855-867.
  29. Park, J.Y., Park, M.R., Kwon, D.N., Kang, M.H., Oh, M., Han, J.W., Cho, S.G., Park, C., Kim, D.K., Song, H., et al. (2011). Alpha 1,3- galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity. J. Biomed. Biotechnol. 2011, 560850.
  30. Park, J.Y., Park, M.R., Bui, H.T., Kwon, D.N., Kang, M.H., Oh, M., Han, J.W., Cho, S.G., Park, C., Shim, H., et al. (2012). alpha1,3- galactosyltransferase deficiency in germ-free miniature pigs increases N-glycolylneuraminic acids as the xenoantigenic determinant in pig-human xenotransplantation. Cell Reprogram 14, 353-363.
  31. Puga Yung, G.L., Li, Y., Borsig, L., Millard, A.L., Karpova, M.B., Zhou, D., and Seebach, J.D. (2012) Complete absence of the alphaGal xenoantigen and isoglobotrihexosylceramide in alpha1,3galactosyltransferase knock-out pigs. Xenotransplantation 19, 196-206.
  32. Roseman, S. (2001). Reflections on glycobiology. J. Biol. Chem. 276, 41527-41542.
  33. Roth, J., Zuber, C., Park, S., Jang, I., Lee, Y., Kysela, K. G., Le Fourn, V., Santimaria, R., Guhl, B., and Cho, J.W. (2010). Protein N-glycosylation, protein folding, and protein quality control. Mol. Cells 30, 497-506.
  34. Sandrin, M.S., and McKenzie, I.F. (1994). Gal alpha (1,3)Gal, the major xenoantigen(s) recognised in pigs by human natural antibodies. Immunol. Rev. 141, 169-190.
  35. Toyoda, M., Ito, H., Matsuno, Y.K., Narimatsu, H., and Kameyama, A. (2008). Quantitative derivatization of sialic acids for the detection of sialoglycans by MALDI MS. Anal. Chem. 80, 5211-5218.
  36. Wada, Y., Azadi, P., Costello, C.E., Dell, A., Dwek, R.A., Geyer, H., Geyer, R., Kakehi, K., Karlsson, N.G., Kato, K., et al. (2007). Comparison of the methods for profiling glycoprotein glycans-- HUPO human disease glycomics/proteome initiative multiinstitutional study. Glycobiology 17, 411-422.
  37. Zhao, Y.Y., Takahashi, M., Gu, J.G., Miyoshi, E., Matsumoto, A., Kitazume, S., and Taniguchi, N. (2008). Functional roles of Nglycans in cell signaling and cell adhesion in cancer. Cancer Sci. 99, 1304-1310.