Figure 1.
MUC5B in human whole saliva sediments faster in the presence of EGCG.
Human whole saliva was incubated with (A) PBS, (B) 0.5 mM EGCG, (C) 1 mM EGCG or (D) 4 mM EGCG and subjected to rate-zonal centrifugation in 10–50% (w/v) sucrose gradients for 1 hour. MUC5B was detected by agarose gel electrophoresis and western blotting with the MAN-5BIII antiserum (insets). Bands were quantified using the Odyssey Imaging system.
Figure 2.
Increased sedimentation rate of purified MUC5B in the presence of 1 mM EGCG.
Rate-zonal centrifugation of MUC5B (A: 200 µg/mL, B: 400 µg/mL, C: 800 µg/mL, D: 1.6 mg/mL) with 1 mM EGCG (grey triangles) or PBS (black circles), performed in 10–35% (w/v) sucrose gradients in PBS. MUC5B was detected by agarose gel electrophoresis and western blotting with the MAN-5BIII antiserum (insets; MUC5B (top panel) and MUC5B+EGCG (bottom panel)). Bands were quantified using the Odyssey Imaging system.
Figure 3.
PTM of polystryrene beads in MUC5B and EGCG solutions.
MSD of 505 nm polystyrene beads in mixtures of MUC5B (A: 100 µg/mL, B: 200 µg/mL, C: 400 µg/mL, D: 800 µg/mL, E: 1.6 mg/mL) with 1 mM EGCG (grey) or PBS (black). MSDs were derived from 15 videos per sample (four samples were measured for each mixture, n = 4). Representative ensemble MSDs are shown. F: MSDs were used to calculate viscosity for MUC5B (black) and MUC5B+EGCG mixtures (grey), (n = 4). The error bars show standard deviation. Parametric paired t-tests were performed to determine signficance; 200, 400, 800 and 1600 µg/mL were statistically significant compared to the control, with p values of 0.0007, 0.0017, 0.0039 and 0.0027 respectively. ** p value 0.001–0.01. *** p value 0.0001–0.001.
Figure 4.
AFM reveals alteration to the MUC5B network by EGCG.
MUC5B at a concentration of 15 µg/mL was imaged with PBS (A–C), 75 µM EGCG (D–F) and 375 µM EGCG (G–I) on APTES-coated mica. Representative images are shown. Scale bars represent 1 µm in all cases. The height scale of the images is 4 nm (A–C), 9 nm (D), 7 nm (E), 10 nm (F), 6 nm (G, I) and 5 nm (H). J: Mean area of aggregates above 1 nm in height. AFM images of MUC5B with PBS (untreated, circles), 75 µM EGCG (treated low-dose, squares) and 375 µM EGCG (treated high-dose, triangles) were quantified using Nanoscope software (n = 10). Particles above a height threshold of 1 nm were identified and their area quantified, the mean is plotted here. The variation seen in the presence of EGCG suggests heterogeneity of aggregation with increasing EGCG concentration. Parametric paired t-tests were performed to determine signficance. Ns = not significant. *** p value 0.0001–0.001.
Figure 5.
Aggregation of MUC5B protein domains by EGCG.
(A) Rate-zonal centrifugation of 380 µg/mL NT5B recombinant protein (D1-D2-D′-D3 domains of MUC5B) in PBS (black circles) or 4 mM EGCG (grey triangles) in 5–20% (w/v) sucrose gradients (n = 2). Sucrose gradients were fractionated into 21 fractions and pelleted material was solubilised in urea. Fractions were run on SDS-PAGE gels and silver stained. NT5B is expressed as a monomer and a dimer. Inset (i) shows the SDS-PAGE gel of the first 11 fractions of NT5B in PBS sucrose gradient, showing all NT5B was contained within the first 11 fractions. Inset (ii) shows the SDS-PAGE gel of fractions 12–21 and the pellet of an NT5B with EGCG gradient, showing that in the presence of EGCG all NT5B is pelleted to the bottom of the tube. (B) Rate-zonal centrifugation of 500 µg/mL CT5B recombinant protein (D4-B-C-CK domains of MUC5B) in PBS (black circles) or 4 mM EGCG (grey triangles) in 5–20% (w/v) sucrose gradients (n = 2). Gradients were fractionated into 19 fractions and pelleted material was solubilised in urea. Fractions were run on SDS-PAGE gels and Coomassie stained. CT5B is expressed as a monomer and a dimer. Inset (i) shows the SDS-PAGE of the first 10 fractions of a sucrose gradient of CT5B in PBS, showing all CT5B was present in the first 5 fractions. Insets (ii) and (iii) show the SDS-PAGE gels of fractions 1–10, and 11–19 and the pellet, respectively, showing that in the presence of EGCG, there is faster sedimentation of CT5B but not full aggregation. Stained SDS-PAGE gels were imaged on a Biorad ChemiDoc MP Imaging System to measure band intensities. Pel. = pelleted material.
Table 1.
Distribution of untreated and EGCG-treated NT5B across the sucrose gradient.
Table 2.
Distribution of untreated and EGCG-treated CT5B across the sucrose gradient.
Figure 6.
MUC5B oligosaccharide-rich regions do not aggregate in the presence of excess EGCG.
MUC5B T-domains were generated by trypsin-digestion and made to 200 µg/mL (A) and 600 µg/mL (B) before rate-zonal centrifugation with PBS (black circles) or 4 mM EGCG (grey triangles), in 5–20% (w/v) sucrose gradients. EGCG was removed from fractions using a HiTrap desalting column, fractions were slot blotted and PAS stained to detect glycoprotein (inset). Blots were scanned using Biorad ChemiDoc MP Imaging System and intensities measured using ImageLab software.
Figure 7.
Aggregation of MUC7 in human whole saliva by EGCG.
Agarose gel electrophoresis of rate-zonal centrifugation fractions of human whole saliva with (A) PBS, (B) 100 µM EGCG, (C) 500 µM EGCG and (D) 1 mM EGCG in 10–50% (w/v) sucrose gradients, probed with EUMUC7a and developed using the Odyssey Imaging system.
Figure 8.
MUC7, purified by isopycnic density gradient centrifugation, interacts with EGCG. Agarose gel electrophoresis of rate-zonal centrifugation fractions of 115 µg/mL purified MUC7 in 5–20% (w/v) sucrose gradients with PBS (A) or 4 mM EGCG (B).
Gels were transferred to nitrocellulose, probed with the EUMUC7a anti-MUC7 antibody and developed using Odyssey Imaging software.
Figure 9.
EC does not cause aggregation of salivary mucins.
Rate-zonal centrifugation of human whole saliva with 1 mM EC (grey triangles) or PBS (black circles), performed in 10–50% (w/v) sucrose gradients in PBS pH 7.4. (A) MUC5B was detected by agarose gel electrophoresis of rate-zonal fractions and western blotting with the MAN-5BIII antiserum (inset; saliva (top panel) and saliva+EC (bottom panel)). (B) MUC7 was detected by agarose gel electrophoresis of rate-zonal fractions and western blotting with the EUMUC7a antiserum (inset; saliva (top panel) and saliva+EC (bottom panel)). Bands were quantified using the Odyssey Imaging system.