Selective bioorthogonal probe for N-glycan hybrid structures

Abstract

Metabolic incorporation of chemically tagged monosaccharides is a facile means of tagging cellular glycoproteins and glycolipids. However, 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 posttranslational modification, with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid and complex. Here we describe the synthesis of 1,3-Pr₂-6-OTs GlcNAlk (MM-JH-1) as a next-generation metabolic chemical reporter for the selective labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labeling with chemically tagged monosaccharides. We then applied this approach to establish that MM-JH-1 is selectively incorporated into hybrid N-glycans. Using this metabolic chemical reporter as a detection tool, we performed imaging and fractionation to define features of the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.

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 shows that the labeling is not O-linked, as MM-JH-1 (detected with FTIC or TAMRA antibody) signal remains unchanged after incubation with NaOH. Assessment of O-GlcNAc (detected with RL2 antibody, MA-1072) was used as a control to ensure successful β-elimination. Graphs showing quantification of signal are to the right of the respective blots (N = 4; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant, error bar represents standard deviation). (C) PNGase F treatment removed the MM-JH-1 labeled signals (detected with FTIC or TAMRA antibody). Loss of total lectin binding proteins (detected with ConA signal) implied that the PNGase F treatment properly removed the N-glycans. Graphs showing quantification of signal 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 incorporated into N-linked glycans. (A) HeLa cells were treated with MM-JH-1 or DMSO and with or without OGT inhibitor OSMI-1 as indicated. Labeling by MM-JH-1 (green, detected with AF 488 staining on confocal images) remained, whereas O-GlcNAc staining (red, detected with RL2 antibody, MA-1076) diminished. Quantification of the images was completed by normalizing mean fluorescent signal to DAPI (blue), and each quantification is shown below the respective image (N = 3; n = 25; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant; N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm and 10 mm for zoomed in images, error bar represents standard deviation). (B) HeLa cells were either treated with tunicamycin, an inhibitor of N-linked glycosylation, or left untreated as indicated, followed by treatment with DMSO or MM-JH-1 for an additional 48 h. Tunicamycin treatment diminished labeling by MM-JH-1 (green, detected with AF 488 staining) shown by confocal imaging. Calnexin (red) was used to assess successful tunicamycin treatment. Quantification of images was done by normalizing mean fluorescent signal to DAPI (blue), and each quantification is shown below the respective image (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 and 10 mm for zoomed in images, error bar represents standard deviation). (C) Tunicamycin treatment diminished MM-JH-1 labeling, shown by western blotting. Loss of MM-JH-1 labeled signal (detected with TRITC or TAMRA antibody) and diminished lectin binding proteins (detected with ConA signal) implied successful inhibition of N-glycan formation. Graphs showing quantifications are also provided (N = 3; An ordinary one-way ANOVA test shows ****p < 0.0001, error bar represents standard deviation).

Figure 3.

The compound MM-JH-1 incorporates into hybrid structure N-glycans. (A) Schematic representation of different types of N-glycan structures, and where inhibitors act. (B) Endo H treatment of immunoblots removed the labeling by MM-JH-1, as indicated by loss of TRITC signal. Assessment of ConA was used as a control to ensure efficacy of Endo H treatment. Quantification is shown to the right of the blots (N = 3; An ordinary one-way ANOVA test shows ****p < 0.0001, error bar represents standard deviation; ns = not significant). (C) Glucosidase inhibitor Castanospermine (CAST) reduced MM-JH-1 labeling (green, detected with AF 488 staining on confocal images). Augmented ConA labeling (red, detected with ConA-Texas red staining on confocal images) indicated the inhibitory activity of CAST. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue), and each quantification is shown below the respective image (N = 3; n = 25; An ordinary one-way ANOVA test shows ****p < 0.0001, **p = 0.0035, *p = 0.0491 or 0.0199, N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm and 10 mm for zoomed in images, error bar represents standard deviation). (D) Mannosidase-I inhibitor 1-Deoxymannojirimycine (DMJM) decreased the signals of labeling by MM-JH-1 (green, detected with AF 488 staining on confocal images). Increased ConA signals (red, detected with ConA-Texas red staining on confocal images) signified the inhibitory activity of DMJM. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue), and each quantification is shown below the respective image (N = 3; n = 25; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant; N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm and 10 mm for zoomed in images, error bar represents standard deviation). (E) Mannosidase-II inhibitor Swainsonine increased the signals of both labeling by MM-JH-1 (green, detected with AF 488 staining on confocal images) and ConA (red, detected with ConA-Texas red staining on confocal images). All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue), and each quantification is shown below of the respective image (N = 3; n = 25; An ordinary one-way ANOVA test shows ****p < 0.0001, ns = not significant, N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm and 10 mm for zoomed in images, error bar represents standard deviation).

Figure 4.

ESI-MS Analysis of MM-JH-1 GlcNAlk-Derivatized Man5 N-Glycan. This figure presents electrospray ionization mass spectrometry (ESI-MS) data for the analysis of N-glycans derived from HeLa cells treated with MM-JH-1 or DMSO. The glycans were released using Endo-H treatment. (A) ESI-MS spectrum profile of the N-glycans. The spectrum is deconvoluted, showing masses of permethylated N-glycans with sodium adducts (M+Na). Proposed structures for the N-glycans and their corresponding MM-JH-1-derivatized forms are depicted next to their respective ions. Masses of the MM-JH-1-derivatized glycans are highlighted in red. The inset shows the Man6 N-glycan (mass 1538.783) and its proposed MM-JH-1-derivatized structure (mass 1990.975). (B) Full MS/MS spectrum of the MM-JH-1-derivatized Man5 N-glycan. The inset shows the structure of the MM-JH-1-derivatized Man5 N-glycan (mass 1990.943) with fragmentation sites observed in the MS/MS spectrum indicated. Green circles represent mannose residues, blue squares represent N-acetylglucosamine residues, and red hexagons represent MM-JH-1 modifications.

Figure 5.

The compound MM-JH-1 was enzymatically added to glycans by MGAT1. (A) siRNA against MGAT1 reduced MGAT1 levels by about 50%. MGAT1 knockdown in HeLa cells reduced the signal of labeling by MM-JH-1 on western blot as detected by TRITC. Quantification is shown beside blots (N = 3; For MGAT1 an ordinary one-way ANOVA test shows ***p = 0.0001; ***p = 0.0003, ns = not significant and for TRITC an unpaired t test shows ****p < 0.0001). (B) MGAT1 siRNA knockdown in HeLa cells reduced the signal of MM-JH-1 (green, detected with AF 488 staining on confocal images) as seen through confocal imaging. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue), and the quantification is shown beside the respective images (N= 3; n =10; An unpaired t-test shows ****p < 0.0001; N = Number of experiment repeat, n = Number of individual cells chosen for quantification of the confocal images, scale bar = 50 mm and 10 mm for zoomed in images, error bar represents standard deviation). (C) MGAT1 cDNA rescue on HEK 293S GnTI⁻ cells results in a return in signal for both MGAT1 (detected with anti-MGAT1 antibody) and MM-JH-1 labeling (detected with anti TRITC antibody) on immunoblot. Representative image is shown from three experimental replicates. (D) Putative binding pose for UDP-MM-JH-1 within the catalytic pocket of human MGAT1. Receptor residues and water molecules located within 3 Å of the ligand are displayed as pink sticks, while the ligand is displayed in green. Hydrogen bonds are reported as black dashed lines, while π-π stacking interactions are illustrated as blue dashed lines. (E) Putative binding pose for UDP-MM-JH-1 within the catalytic pocket of human MGAT2. Receptor residues and water molecules located within 3 Å of the ligand are displayed as blue sticks, while the ligand is displayed in green. Hydrogen bonds are reported as black dashed lines, while π-π stacking interactions are illustrated as blue dashed lines.

Figure 6.

The nucleolar protein Fibrillarin is modified by MM-JH-1. (A) Change in the colocalization between fibrillarin or nucleolin signal and MM-JH-1 signal with Actinomycin D treatment. Actinomycin D treatment was used to disrupt the nucleolus. Colocalization of fibrillarin (red, detected with anti-nucleolar marker fibrillarin antibody) and nucleolin signal (red, detected with anti-nucleolin antibody) with MM-JH-1 (green, detected with AF 488 signal on confocal images) was examined. Colocalization was determined by Pearson’s R value as indicated to the right of images. One representative image is shown from three experimental replicates. Scale bar = 50 mm and 10 mm for zoomed in images. (B) After performing a click reaction with a biotin-alkyne, streptavidin was used on lysates to pulldown MM-JH-1 labeled proteins. 5% of input, flowthrough from an IgG elution buffer (to assess non-specific interactions), and the Biotin/Streptavidin pulldown were all run 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 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). (C) Cytoplasmic fibrillarin and increased MM-JH-1 expression were detected with Brefeldin A (BFA) treatment (N= 3).