Abstract
Anti-carbohydrate monoclonal antibodies (mAbs) are very useful in the functional analysis of complex carbohydrates in vivo. However, such mAbs are difficult to generate, largely because a wide variety of complex carbohydrates is intrinsically expressed in mice and rats and because the antigenicities of glycans are generally poor. In this chapter, I describe an efficient method for generating anti-carbohydrate mAbs using glycan-synthesizing enzyme-knockout mice in which the glycan structures formed by the missing enzymes should be highly antigenic. As an application of this method, I describe the generation of anti-sulfated glycan mAbs using sulfotransferase-deficient mice and the immunohistochemical detection of sulfated glycans involved in lymphocyte homing in both humans and mice.
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References
Kawashima H, Petryniak B, Hiraoka N, Mitoma J, Huckaby V, Nakayama J, Uchimura K, Kadomatsu K, Muramatsu T, Lowe JB, Fukuda M (2005) N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules. Nat Immunol 6:1096–1104
Uchimura K, Gauguet JM, Singer MS, Tsay D, Kannagi R, Muramatsu T, von Andrian UH, Rosen SD (2005) A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules. Nat Immunol 6:1105–1113
Lee J, Park SH, Stanley P (2002) Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III. Glycoconj J 19:211–219
Tahara H, Ide K, Basnet NB, Tanaka Y, Matsuda H, Takematsu H, Kozutsumi Y, Ohdan H (2010) Immunological property of antibodies against N-glycolylneuraminic acid epitopes in cytidine monophospho-N-acetylneuraminic acid hydroxylase-deficient mice. J Immunol 184:3269–3275
Hirakawa J, Tsuboi K, Sato K, Kobayashi M, Watanabe S, Takakura A, Imai Y, Ito Y, Fukuda M, Kawashima H (2010) Novel anti-carbohydrate antibodies reveal the cooperative function of sulfated N- and O-glycans in lymphocyte homing. J Biol Chem 285:40864–40878
Arata-Kawai H, Singer MS, Bistrup A, Zante A, Wang YQ, Ito Y, Bao X, Hemmerich S, Fukuda M, Rosen SD (2011) Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs. Am J Pathol 178:423–433
Mitoma J, Bao X, Petryanik B, Schaerli P, Gauguet JM, Yu SY, Kawashima H, Saito H, Ohtsubo K, Marth JD, Khoo KH, von Andrian UH, Lowe JB, Fukuda M (2007) Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment. Nat Immunol 8:409–418
Homeister JW, Thall AD, Petryniak B, Maly P, Rogers CE, Smith PL, Kelly RJ, Gersten KM, Askari SW, Cheng G, Smithson G, Marks RM, Misra AK, Hindsgaul O, von Andrian UH, Lowe JB (2001) The α(1, 3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing. Immunity 15:115–126
Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M (2001) Novel sulfated lymphocyte homing receptors and their control by a Core1 extension β1,3-N-acetylglucosaminyltransferase. Cell 105: 957–969
Kawano T, Koyama S, Takematsu H, Kozutsumi Y, Kawasaki H, Kawashima S, Kawasaki T, Suzuki A (1995) Molecular cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase. Regulation of species- and tissue-specific expression of N-glycolylneuraminic acid. J Biol Chem 270:16458–16463
Mitoma J, Miyazaki T, Sutton-Smith M, Suzuki M, Saito H, Yeh JC, Kawano T, Hindsgaul O, Seeberger PH, Panico M, Haslam SM, Morris HR, Cummings RD, Dell A, Fukuda M (2009) The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells. Glycoconj J 26:511–523
Kannagi R, Ohmori K, Kimura N (2009) Anti-oligosaccharide antibodies as tools for studying sulfated sialoglycoconjugate ligands for siglecs and selectins. Glycoconj J 26:923–928
Acknowledgments
I would like to thank Drs. Jotaro Hirakawa (University of Shizuoka, School of Pharmaceutical Sciences), Minoru Fukuda (Sanford-Burnham Medical Research Institute), and Motohiro Kobayashi (Shinshu University, School of Medicine) for collaboration. This work was supported in part by Grants-in-Aid for Scientific Research, Category (B) and Grants-in-Aid for Scientific Research on Priority Areas, Dynamics of Extracellular Environments, from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21390023 and 20057022, respectively).
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Kawashima, H. (2013). Generation of Anti-sulfated Glycan Antibodies Using Sulfotransferase-Deficient Mice. In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_5
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DOI: https://doi.org/10.1007/978-1-62703-465-4_5
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