Abstract
Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation, but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the native protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP (2011) Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells. J Biol Chem 286(8):6780–6790. doi:10.1074/jbc.M110.179002
Dias-Baruffi M, Stowell SR, Song SC, Arthur CM, Cho M, Rodrigues LC, Montes MA, Rossi MA, James JA, McEver RP, Cummings RD (2009) Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle. Glycobiology 20(5):507–520
Cooper DN, Barondes SH (1990) Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism. J Cell Biol 110(5):1681–1691
Arthur CM, Cummings RD, Stowell SR (2014) Using glycan microarrays to understand immunity. Curr Opin Chem Biol 18C:55–61. doi:10.1016/j.cbpa.2013.12.017
Stowell SR, Cho M, Feasley CL, Arthur CM, Song X, Colucci JK, Karmakar S, Mehta P, Dias-Baruffi M, McEver RP, Cummings RD (2009) Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. J Biol Chem 284(8):4989–4999. doi:10.1074/jbc.M808925200
Cerri DG, Rodrigues LC, Stowell SR, Araujo DD, Coelho MC, Oliveira SR, Bizario JC, Cummings RD, Dias-Baruffi M, Costa MC (2008) Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1. Glycobiology 18(11):842–850
Cho M, Cummings RD (1995) Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis. J Biol Chem 270(10):5207–5212
Stowell SR, Arthur CM, McBride R, Berger O, Razi N, Heimburg-Molinaro J, Rodrigues JP, Noll AJ, von Gunten S, Smith DF, Knirel YA, Paulson JC, Cummings RD (2014) Microbial glycan microarrays define key features of host-microbial interactions. Nat Chem Biol 10:470–476
Stowell SR, Arthur CM, Dias-Baruffi M, Rodrigues LC, Gourdine JP, Heimburg-Molinaro J, Ju T, Molinaro RJ, Rivera-Marrero C, Xia B, Smith DF, Cummings RD (2010) Innate immune lectins kill bacteria expressing blood group antigen. Nat Med 16(3):295–301. doi:10.1038/nm.2103
van Kooyk Y, Rabinovich GA (2008) Protein-glycan interactions in the control of innate and adaptive immune responses. Nat Immunol 9(6):593–601. doi:10.1038/ni.f.203
Tracey BM, Feizi T, Abbott WM, Carruthers RA, Green BN, Lawson AM (1992) Subunit molecular mass assignment of 14,654 Da to the soluble beta-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation. J Biol Chem 267(15):10342–10347
Hirabayashi J, Kasai K (1991) Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin. J Biol Chem 266(35):23648–23653
Cho M, Cummings RD (1995) Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization. J Biol Chem 270(10):5198–5206
Stowell SR, Qian Y, Karmakar S, Koyama NS, Dias-Baruffi M, Leffler H, McEver RP, Cummings RD (2008) Differential roles of galectin-1 and galectin-3 in regulating leukocyte viability and cytokine secretion. J Immunol 180(5):3091–3102
Inagaki Y, Sohma Y, Horie H, Nozawa R, Kadoya T (2000) Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties. Eur J Biochem/FEBS 267(10):2955–2964
Cerliani JP, Stowell SR, Mascanfroni ID, Arthur CM, Cummings RD, Rabinovich GA (2011) Expanding the universe of cytokines and pattern recognition receptors: galectins and glycans in innate immunity. J Clin Immunol 31(1):10–21. doi:10.1007/s10875-010-9494-2
Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, Rabinovich GA (2007) Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death. Nat Immunol 8(8):825–834. doi:10.1038/ni1482
Perillo NL, Pace KE, Seilhamer JJ, Baum LG (1995) Apoptosis of T cells mediated by galectin-1. Nature 378(6558):736–739. doi:10.1038/378736a0
Tartier L, McCarey YL, Biaglow JE, Kochevar IE, Held KD (2000) Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria. Cell Death Differ 7(10):1002–1010. doi:10.1038/sj.cdd.4400726
Braakman I, Helenius J, Helenius A (1992) Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J 11(5):1717–1722
Stowell SR, Karmakar S, Stowell CJ, Dias-Baruffi M, McEver RP, Cummings RD (2007) Human galectin-1, -2, and -4 induce surface exposure of phosphatidylserine in activated human neutrophils but not in activated T cells. Blood 109(1):219–227
Go YM, Jones DP (2013) Thiol/disulfide redox states in signaling and sensing. Crit Rev Biochem Mol Biol 48(2):173–181. doi:10.3109/10409238.2013.764840
Clerch LB, Whitney P, Hass M, Brew K, Miller T, Werner R, Massaro D (1988) Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin. Biochemistry 27(2):692–699
Acknowledgments
This work was supported in part by grants from the National Blood Foundation, American Society of Hematology and Hemophilia of Georgia to S.R.S.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Stowell, S.R., Arthur, C.M., Cummings, R.D., Feasley, C.L. (2015). Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation. In: Stowell, S., Cummings, R. (eds) Galectins. Methods in Molecular Biology, vol 1207. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1396-1_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1396-1_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1395-4
Online ISBN: 978-1-4939-1396-1
eBook Packages: Springer Protocols