Selective bioorthogonal probe for N-glycan hybrid structures

Abstract

Metabolic incorporation of chemically tagged monosaccharides is a facile means of labelling cellular glycoprotein and glycolipids. Yet, since the monosaccharide precursors are often shared by several pathways, selectivity has been difficult to attain. For example, N-linked glycosylation is a chemically complex, and ubiquitous post translational modification with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid, and complex. Here we describe synthesis of 1,3-Pr₂-6-OTs GlcNAlk as a next generation metabolic chemical reporter (MCR) for the specific labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labelling with chemically tagged monosaccharides. We then applied this approach to establish that 1,3-Pr₂-6-OTs GlcNAlk is specifically incorporated into hybrid N-glycans. Using this MCR as a detection tool, we carried out imaging experiments to define the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.

Keywords: 1,3-Pr2-6-OTs GlcNAlk; Bioorthogonal chemistry; Enzymatic labeling; Fibrillarin nuclear protein; Hybrid N-Glycan.

Figure 1.

Compound MM-JH-1 was specific to N-linked glycosylation (A) Chemical synthesis of the title compound MM-JH-1. (b) β-elimination study showing the labeling are not O-linked, with MM-JH-1 (detected with RL2 antibody) signal remaining after incubation with NaOH. Assessment of O-GlcNAc (detected with RL2 antibody) was used as a control to ensure successful β-elimination (N = 4; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant). (c) PNGase F treatment removed the MM-JH-1 labeled signals. RL2 signal remained unchanged while ConA signal decreased, indicating PNGase F properly removed N-Linked glycans. Graph showing quantifications are to the right of the respective blots (N = 3; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant, error bar represents standard deviation).

Figure 2.

The compound MM-JH-1 was enzymatically added to the N-linked glycans. (a) Cells were treated with or without MM-JH-1 and with or without OGT inhibitor OSMI-1 as indicated. Whereas O-GlcNAc staining (red) diminished, there was no effect on labeling by this compound (green, AF 488). Quantification of images were done by normalizing mean fluorescent signal to DAPI (blue) are shown to the right (b) Cells were treated or left untreated with Tunicamycin, an inhibitor of N-linked glycosylation, as indicated. Treatment diminished labeling by MM-JH-1 (green, AF 488) shown by confocal imaging. Calnexin (red) was used to assess successful tunicamycin treatment. Quantification of images were done by normalizing mean fluorescent signal to DAPI (blue) are shown to the right. Quantification of images were done by normalizing mean fluorescent signal to DAPI (blue) are shown under the images. (N = 3; n = 10; An ordinary one-way ANOVA test shows ****p < 0.0001, ***p = 0.0001; N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm). (c) Increasing amounts of Tunicamycin (as indicated) inhibited labeling by MM-JH-1 shown by western blotting. (N = 3; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant, error bar represents standard deviation).

Figure 3.

The compound MM-JH-1 incorporates into hybrid structures of N-glycans. (a) Picture indicating types of N-glycan structures and where inhibitors act. (b) Endo H treatment of immunoblots removed the labeling by the compound MM-JH-1, as indicated by TAMRA. Assessment of O-GlcNAc and ConA were used as controls to ensure efficacy of Endo H reaction with quantification shown to the right of blots. (c) Glucosidase inhibitor Castanospermine (CAST) had no effect on the labeling by MM-JH-1 (green). (d) Mannosidase-I inhibitor 1-Deoxymannojirimycine (DMJM) reduced the signals of labeling by MM-JH-1 (green). (e) Mannosidase-II inhibitor Swainsonine increased the signals of labeling by MM-JH-1 (green). All quantification of images were done by normalizing mean fluorescent signal to DAPI (blue) are shown to the right of the respective images. (N = 3; n = 10; An ordinary one-way ANOVA test shows ****p < 0.0001, ***p = 0.0001, ns = not significant, scale bar = 50 mm, error bar represents standard deviation).

Figure 4.

The compound MM-JH-1 was added to glycans by MGAT1. (a) siRNA against MGAT1 reduced MGAT1 levels by about 50%. MGAT1 knockdown reduced the signal of labeling by MM-JH-1 on western blotting as detected by TAMRA. Quantification is shown below blots (N = 3; For MGAT1 an ordinary one-way ANOVA test shows ****p < 0.0001; ***p = 0.0003, ns = not significant and for TAMRA an unpaired t test shows ***p = 0.0001). (b) MGAT1 knockdown reduced the signal of labeling by MM-JH-1 (green) on confocal imaging. Quantification is shown to the right of images (N= 3; n =10; An an unpaired t-test shows ****p < 0.0001; ***p = 0.0003, ns = not significant, scale bar = 10 mm, error bar represents standard deviation). (c) Schematic representation of addition of MM-JH-1 by MGAT1 to the core of oligo mannose structures and the resulting hybrid structures.

Figure 5.

The nucleolar protein Fibrillarin is modified by the MM-JH-1 compound. (a) Colocalization of fibrillarin and nucleolin signal (red) with MM-JH-1 (green). Colocalization was determined by Pearson’s R value as indicated to the right of images. (b) After performing a click reaction with a biotin-alkyne, Streptavidin was used on lysates to pulldown MM-JH-1 labelled proteins. 5% of input, flowthrough from an IgG elution buffer (to assess non-specific interactions), and the Biotin/Streptavidin pulldown were all run out on a gel, transferred to a membrane and immunoblotted using a fibrillarin antibody (left panel). A band indicating fibrillarin is visible in the 5% input and biotin-pull down lanes. These lysates were treated with Endo H, run out on a gel and immunoblotted for fibrillarin. In this case fibrillarin signal is lost in the biotin pull down lane indicating that Endo H cleaved the sugar from fibrillarin. (N= 3; n =10, scale bar = 10 mm)