Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation
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.
Key wordsAlkylation Galectin Mass spectrometry Oxidation Reducing agents
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.
- 1.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 PubMedCrossRefPubMedCentralGoogle Scholar
- 2.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):507–520CrossRefGoogle Scholar
- 5.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 PubMedCrossRefPubMedCentralGoogle Scholar
- 8.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 10:470–476PubMedCrossRefPubMedCentralGoogle Scholar
- 9.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 PubMedCrossRefPubMedCentralGoogle Scholar
- 11.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):10342–10347PubMedGoogle Scholar
- 17.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 PubMedCrossRefGoogle Scholar