Journal: bioRxiv

Article Title: Intact and single-molecule analysis of heparan sulfate

doi: 10.64898/2026.06.26.734651

Figure Lengend Snippet: a. Schematic of end-selective mono-labeling of intact heparan sulfate (HS). PmHS2 installs a single azide-modified GlcNAc from UDP-GlcNAz onto GlcA-capped non-reducing ends (NREs); GlcNAc-capped NREs are first repaired by PmHS2-mediated addition of GlcA from UDP-GlcA. The reducing end (RE) is chemically functionalized by hydroxylamination using an aminooxy-azide linker. Sugar symbols follow SNFG notation , ; red circles indicate potential sulfation sites. b. Conventional DEAE anion-exchange purification requires high-salt elution, desalting, and lyophilization. Silica column-based purification of HS uses guanidinium-mediated binding followed by rapid elution in water. c. Recovery of representative glycosaminoglycans after silica purification, quantified by Alcian Blue staining. Analytes include cell-derived HS of varying sulfation levels (rHS01, rHS05, rHS09), heparin (HP), chondroitin sulfate (CS-A, CS-D), and hyaluronic acid (HA). Data from . n = 3; bars indicate mean ± s.d. d. Recovery of [ 3 H]heparin after silica purification. [ 3 H]heparin was spiked into increasing amounts of unlabeled heparin, purified over a silica column, and eluate fractions were quantified by liquid scintillation counting. Curve represents a nonlinear regression fit to a one-site total binding model (GraphPad Prism). n = 2; bars indicate mean ± s.d. e. Wild-type (rHS01) and lowly-sulfated (rHS05) HS treated with UDP-GlcA with (+) or without (-) PmHS2 before UDP-GlcNAz labeling and IR800 detection. Data from . n = 3; bars indicate mean ± s.d. Percentages indicate the increase in IR800 fluorescence after NRE repair. f. Comparison of end-selective labeling of rHS01 and rHS05 assessed by SDS-PAGE with IR800 detection. HS chains were labeled enzymatically at the NRE or chemically at the RE. Right, quantification of IR800 fluorescence from labelled HS species. Data from . n = 3; bars indicate mean ± s.d. Percentages indicate NRE labeling signal relative to the corresponding RE-labeled sample.

Article Snippet: For PmHS2-mediated labeling, heparan sulfate was combined in a final volume of 20 μL containing 40 mM Tris-HCl (pH 7.5, T1075, Teknova), 4 mM MnCl 2 , 4 mM MgCl 2 , 100 μM UDP-GlcNAz (HY-145934A, MedChemExpress) or 100 μM UDP-GlcA (U6751, Sigma), and 450 μg/mL of PmHS2, using a modified version of previously reported reaction conditions .

Techniques: Labeling, Modification, Purification, Lyophilization, Binding Assay, Staining, Derivative Assay, Fluorescence, Comparison, SDS Page

Journal: bioRxiv

Article Title: Intact and single-molecule analysis of heparan sulfate

doi: 10.64898/2026.06.26.734651

Figure Lengend Snippet: a. Schematic of chemoenzymatic mono-labeling of GlcA-capped non-reducing ends (NREs) on native heparan sulfate chains using UDP-GlcNAz only, followed by copper-free click (SPAAC) with IR800-DBCO. b. SDS-PAGE analysis of cell-derived heparan sulfate (rHS01) labeled with UDP-GlcNAz by either PmHS2 or EXT1/2. Reactions were incubated for 1 hour using different enzyme concentrations (see Methods). The gel was resolved by SDS-PAGE and imaged using infrared fluorescence detection. c. Dependence of mono-NRE labeling on the presence of heparan sulfate, PmHS2, and UDP-GlcNAz. The gel was resolved and imaged as in b . d. Mono-NRE-labeled heparan sulfate with or without in vitro digestion by heparin lyases I, II, and III, and with or without silica column purification. The gel was resolved and imaged as in b . e. Time-course of PmHS2-mediated mono-NRE labeling with UDP-GlcNAz only, with IR800 fluorescence quantified from the indicated gel region. The gel was resolved and imaged as in b . Data were fitted to a one-phase exponential decay model using GraphPad Prism (mean ± s.d., n = 3). The negative control lacked PmHS2 but included all other reaction components. f. Mono-NRE-labeling using UDP-GlcA only extension (“NRE repair”), followed by UDP-GlcNAz-only labeling of naturally sulfated (rHS01) and minimally sulfated (rHS05) heparan sulfate. The gel was resolved and imaged as in b .

Article Snippet: For PmHS2-mediated labeling, heparan sulfate was combined in a final volume of 20 μL containing 40 mM Tris-HCl (pH 7.5, T1075, Teknova), 4 mM MnCl 2 , 4 mM MgCl 2 , 100 μM UDP-GlcNAz (HY-145934A, MedChemExpress) or 100 μM UDP-GlcA (U6751, Sigma), and 450 μg/mL of PmHS2, using a modified version of previously reported reaction conditions .

Techniques: Labeling, SDS Page, Derivative Assay, Incubation, Fluorescence, In Vitro, Purification, Negative Control

Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: A, Schematic diagram of the hexosamine biosynthetic pathway (HBP) from glycolysis to UDP-GlcNAc. B–C , IPI-2I cells were infected with PDCoV (MOI = 2) and harvested at the indicated time points for HBP flux analysis. (B) Protein levels of GFAT, AGX, OGT, PDCoV N, and cellular O-GlcNAcylation, assessed by western blot. Ratios below the images represent the protein levels of GFAT and AGX relative to β-actin, as quantified by the ImageJ software. (C) Intracellular UDP-GlcNAc content, measured by ELISA. D-I, IPI‑2I cells were subjected to the indicated treatments, infected with PDCoV (MOI = 2), and harvested at 12 h post-infection (hpi) for viral replication analysis. (D) Cells supplemented with or without UDP‑GlcNAc (32 mM). Ratios below the images represent the protein levels of PDCoV relative to β-actin, as quantified by the ImageJ software. (E) Cells treated with the O‑GlcNAcylation agonist MK‑8719 (10 μM) or inhibitor OSMI‑1 (50 μM). (F) Wild-type versus OGT knockout (KO) IPI-2I cells. Ratios below the images represent the protein levels of PDCoV relative to β-actin, as quantified by the ImageJ software. (G–H) Wild‑type and OGT (KO) IPI-2I cells supplemented with or without UDP‑GlcNAc. (I) OGT (KO) IPI-2I cells overexpressing OGT or catalytically inactive OGT‑DP, with UDP‑GlcNAc supplementation. β-actin and GAPDH served as internal controls for western blot and RT-qPCR, respectively. Data in panels C, D, E, F, G, H, and I represent mean ± s.d. (n = 3). Statistical significance was determined by two-tailed Student’s t-test; * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Knock-Out, Quantitative RT-PCR, Two Tailed Test

Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: A-B, HBP flux in IPI-2I cells infected with PEDV, TGEV, or SADS-CoV at indicated time points: (A) Protein levels of GFAT, AGX, OGT, viral N protein, and global O-GlcNAcylation, assessed by western blot. Ratios below the images represent the protein levels of GFAT and AGX relative to β-actin, as quantified by the ImageJ software. (B) Intracellular UDP-GlcNAc content, measured by ELISA. C, Viral replication of PEDV, TGEV, or SADS-CoV in IPI-2I cells supplemented with or without UDP-GlcNAc. D , Viral replication of PEDV, TGEV, or SADS-CoV in IPI-2I cells treated with or without the O-GlcNAcylation agonist MK-8719. E-F , Viral replication of PEDV, TGEV, or SADS-CoV in wild-type ( E ) and RNF166 (KO) (F) IPI-2I cells overexpressing RNF166 or RNF166-T157A. G-I , Western blot analysis of the degradation of PEDV M protein (G) , TGEV M protein (H) , SADS-CoV M protein ( I ) and their corresponding mutants (PEDV M-K202R, TGEV M-K239R, SADS-CoV M-K246R) in HEK293T cells transfected with FLAG-RNF166 or FLAG-RNF166-T157A. Ratios below the images represent the protein levels of HA relative to β-actin, as quantified by the ImageJ software. Data in panels B–F represent mean ± s.d. (n = 3). Statistical significance was determined by two-tailed Student’s t-test; * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: PLOS Pathogens

Article Title: O-GlcNAcylation licenses RNF166 to degrade the M protein of porcine coronaviruses

doi: 10.1371/journal.ppat.1014301

Figure Lengend Snippet: Using PDCoV as a representative example, in response to porcine coronavirus infection, host cells enhance the HBP flux, leading to UDP-GlcNAc accumulation. Concurrently, OGT translocates to the cytoplasm and catalyzes the O-GlcNAcylation of RNF166 at T157. This modification licenses RNF166 to mediate the degradation of the PDCoV M protein, while it does not alter the ability of RNF166 to induce interferon production. This mechanism is highly conserved across porcine enteric coronaviruses.

Article Snippet: UDP-GlcNAc (HY-112174) was purchased from MedChemExpress, China.

Techniques: Infection, Modification