High-efficiency labeling of sialylated glycoproteins on living cells

Abstract

We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell-surface sialylated glycoproteins while maintaining high cell viability.

Figure 1

Labeling cell surface glycoproteins by aniline-catalyzed oxime ligation. (a) Scheme for aminooxy-biotin labeling of aldehydes on cell surface sialic acids introduced by periodate oxidation (top panel) or metabolically labeling with aldehyde-substituted NeuAc (^BANeuAc) (bottom panel). (b) Aniline increases labeling with aminooxy-biotin. BJA-B K88 cells subjected to periodate oxidation (1 mM at 4° C for 30 minutes) were reacted with 100 µM aminooxy-biotin in the presence or absence of 10 mM aniline (pH 6.7, at 4 °C for 90 minutes). Incorporated biotin was detected by flow cytometry with DTAF-streptavidin. Control refers to untreated BJA-B K88 cells. P: periodate, BO: aminooxy-biotin, A: aniline (c) Comparison of aminooxy-biotin labeling of aldehydes introduced by periodate or culture with ^BANeuAc. BJA-B K88 and sialic acid deficient BJA-B K20 cells were cultured in serum-free medium with or without NeuAc or ^BANeuAc. Cells were subjected to ligation with aminooxy-biotin ±10 mM aniline, stained with DTAF-streptavidin and subjected to flow cytometry. Values represent the mean and s.d. (n=3). MCF: Mean Channel Fluorescence. (d) One-pot labeling of BJA-B K20 cells. Cells were subjected to one-pot or sequential labeling of surface sialic acids. For the one-pot reaction cells were incubated with 1 mM NaIO₄ and 250 µM aminooxy-biotin with 10 mM aniline at pH 6.7 at 4 °C for 30 minutes. The sequential reaction involved periodate oxidation at pH 7.4 for 30 minutes, followed by the aniline-catalyzed oxime ligation at pH 6.7 for 30 minutes. Results in b–d are representative of 2 or more experiments.

Figure 2

Efficient biotinylation of cell surface glycoproteins using PAL. (a) PAL uniformly labels sialylated glycoproteins. BJA-B K20 cells with ³H-NeuAc labeled glycoproteins were subjected to PAL with 250 µM aminooxy-biotin. The efficiency of PAL was assessed by fluorography of sialylated proteins in cell lysates (Total) and streptavidin pull-downs (IP) resolved by gel electrophoresis. (b) Quantitation of biotin-labeled CD45 from PAL labeled cells. BJA-B K20 cells were subjected to oxime ligation using PAL followed by quantitative anti-CD45 and streptavidin pull downs, and immunoblotting with anti-CD45 antibody. (c) Confocal microscopy of PAL labeled cells. Untreated and periodate treated (1 mM at 4 °C for 5 min) CHO cells were subjected to ligation with aminooxy-biotin (100 µM) ± 10 mM aniline at 4 °C for 90 min. Cells were permeabilized and stained with DTAF-streptavidin (green). Nuclei were stained with DAPI (blue). (d) Endocytosis of PAL labeled proteins following warming to 37 °C. CHO cells, labeled by PAL at 4 °C, were returned to culture medium at 37 °C for the indicated time period (0 or 1 hour), then permeabilized and stained with DTAF-streptavidin (green), and anti-EEA (endosomes) or anti-UH3 (lysosomes) antibody followed by anti-mouse IgG-AF555 (red). Images were obtained by confocal microscopy. Scale bars, 15 µm (c) and 7.5 µm (d).