Fig 2: O-GlcNAcylated HSP27 is a better chaperone against α-synuclein amyloid aggregation.a) Unmodified and differentially O-GlcNAcylated versions of HSP27 were retrosynthetically deconstructed into a recombinant protein thioester and peptides prepared by solid phase peptide synthesis. b) α-Synuclein alone (50 μM) or in the presence of HSP27 or the indicated O-GlcNAcylated HSP27 proteins (1 μM) was subjected to aggregation conditions (agitation at 37 °C). After different lengths of time, aliquots were removed and analyzed by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows fold change in fluorescence compared with α-synuclein alone at t = 0 h. Results are mean ±SEM of n=3 independent experiments. Statistical significance was determined using a one-way ANOVA test followed by Dunnet’s test (α-synuclein alone or plus HSP27 versus O-GlcNAcylated proteins). c) The same reactions were analyzed by TEM after 168 h. d) The same reactions were subjected to the indicated concentrations of proteinase-K (PK) for 30 min before separation by SDS-PAGE and visualization by Coomassie staining. The persistence of bands correlates with the amount of amyloid formation.

Fig 3: ALR-S decreases ROS generation during platelet activation. (A) Platelet intracellular ROS level was determined using flow cytometry. Platelets loaded with H2DCFDA (50 µmol/l) were incubated with ALR-S or vehicle for 10 min and then stimulated with CRP (2 µg/ml), thrombin (0.025 U/ml), or ADP (10 µM) for 5 min. *P<0.05 and **P<0.01 vs. control. (B) Western blotting was performed to analyze the effects of ALR-S on phosphorylated ERK1/2, p38 and HSP27 during platelet activation in an aggregometer. (C) Representative aggregation traces of washed platelets in response to collagen, thrombin or ADP. Washed platelets were preincubated with ALR-S (0, 20, 60 and 200 µg/ml), respectively. H2O2 (20 µM) was added before aggregation was started, and traces were recorded by a Chrono-log aggregometer under stirring. ALR-S, saccharides from Arctium lappa L. root; p38, p38 mitogen-activated protein kinase; HSP27, heat shock protein 27; Col, collagen; Thr, thrombin; MFI, mean fluorescence intensity; ROS, reactive oxygen species; CRP, collagen-related peptide.

Fig 4: Global O-GlcNAc is lower in Alzheimer’s disease but is increased or maintained on HSP27 and αBC, respectively.a) Overall O-GlcNAc is lower in Alzheimer’s disease. Brain samples (Brodmann area 7) from Alzheimer’s patients and age matched controls were analyzed by dot blotting with a pan anti-O-GlcNAc antibody (RL2). RL2 staining was normalized to loading (Ponceau stain) and quantitated. b) HSP27 O-GlcNAc modification is increased in Alzheimer’s disease. O-GlcNAc modified proteins were enriched from Brain samples (Brodmann area 7) from Alzheimer’s patients and age matched controls using chemoenzymatic labeling and analyzed by Western blotting against HSP27. Biotin-IP signal was normalized to input levels and quantitated. c) αBC O-GlcNAc modification is maintained in Alzheimer’s disease. O-GlcNAc modified proteins were enriched from Brain samples (Brodmann area 7) from Alzheimer’s patients and age matched controls using chemoenzymatic labeling and analyzed by Western blotting against αBC. Biotin-IP signal was normalized to input levels and quantitated. In all panels, results are mean ±SEM of n=8 biologically independent samples. Statistical significance was determined using a two-tailed Welch’s t-test.

Fig 5: Additional TEM images of α-synuclein/HSP27 aggregation.Larger format and additional TEM images corresponding to Figure 2c. The images are consistent between all three experimental replicates.