Abstract
The sialome or display of sialic acids on the surface of human immune cells can vary according to immune response and activation state. Here, human peripheral blood mononuclear cells (PBMCs) were isolated and activated with anti-CD3 antibody and the cell surface sialome was quantified using a combination of click chemistry, confocal microscopy and flow cytometry techniques. Carbohydrate click chemistry was used to detect and measure the incorporation of an azido-m65odified sialic acid precursor molecule, N-acetylmannosamine (ManNaz) sugar into the PBMC surface sialome. Incorporation of sialic acid into the PBMC glycocalyx was visualized using copper-catalyzed click conjugation of Alexa 488 alkyne and confocal microscopy and further quantified using flow cytometry. The use of these methods indicate that regulating the sialome content on the surface of activated immune cells may be monitored during immunomodulatory responses and anti-inflammatory therapies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Boligan KF et al (2015) Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense. Cell Mol Life Sci 72(7):1231–1248
Zhuo Y, Bellis SL (2011) Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function. J Biol Chem 286(8):5935–5941
Varki A, Angata T (2006) Siglecs—the major subfamily of I-type lectins. Glycobiology 16(1):1R–27R
Xiao H et al (2016) Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc Natl Acad Sci U S A 113(37):10304–10309
Pfeifle R et al (2017) Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease. Nat Immunol 18:104–113
Ohmi Y et al (2016) Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis. Nat Commun 7:11205
Toscano MA et al (2007) Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death. Nat Immunol 8(8):825–834
Clark MC, Baum LG (2012) T cells modulate glycans on CD43 and CD45 during development and activation, signal regulation, and survival. Ann N Y Acad Sci 1253:58–67
Speers AE, Cravatt BF (2004) Profiling enzyme activities in vivo using click chemistry methods. Chem Biol 11(4):535–546
Baskin JM et al (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A 104(43):16793–16797
Zaro BW, Bateman LA, Pratt MR (2011) Robust in-gel fluorescence detection of mucin-type O-linked glycosylation. Bioorg Med Chem Lett 21(17):5062–5066
Laughlin ST, Bertozzi CR (2007) Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation. Nat Protoc 2(11):2930–2944
Saxon E et al (2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J Am Chem Soc 124(50):14893–14902
Marshall A, Lichtman TJK, Seligsohn U, Kaushansky K, Prchal JT (2011) Williams hematology, 8th edn. McGraw-Hill, Pennsylvania
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
O’Farrell, L.K., Fraser, A.D., Davey, G.P. (2022). Monitoring the Sialome on Human Immune Cells. In: Davey, G.P. (eds) Glycosylation. Methods in Molecular Biology, vol 2370. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1685-7_17
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1685-7_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1684-0
Online ISBN: 978-1-0716-1685-7
eBook Packages: Springer Protocols