Figure 1.
Schematic structures of MUC7 (A) and MUC5B (B), and epitopes recognized by anti-MUC7 and anti-MUC5B antibodies.
MUC7 and MUC5B are heavily glycosylated by oligosaccharide side chains. Positions of putative N- and O-glycosylation sites, and C-mannosylation sites are marked. O-linked sugars possess terminally located sialic acid residues that were omitted for simplicity. EU-MUC7a, a mouse monoclonal antibody recognizes the amino acid sequence EGRERDHELRHRRHHHQ, located in the N-terminal domain (amino acids 21–37) of MUC7. EU-MUC5Bb (IgG1 subclass) is a mouse monoclonal antibody which recognizes amino acid sequence RNREQVGKFKMC, located in four of the cysteine-rich domains of the tandem repeat of MUC5B (amino acids 2388–2399, 2917–2928, 3615–3626, 4144–4155). The figures were drawn based on the information in Uniprot (accession number Q9HC84 and Q8TAX7).
Figure 2.
Analysis of salivary protein degradation.
Saliva was incubated for up to 9 hours at 37°C, followed by SDS-PAGE. Resolved samples were stained for proteins (A), glycoproteins (B), or incubated with anti-MUC7 (C) and anti-MUC5B (D) antibodies. Arrows in figure C and D indicate the positions of MUC7 and MUC5B. Migration positions of molecular weight standard are shown on the left. In figure (E), the results of glycoprotein staining and Western blotting were analyzed densitometrically, and plotted in a semi-log scale. The linear regression curves and half-life times were then calculated. (•): MUC5B (estimated by glycoprotein staining); (○): MUC5B (estimated by Western blotting); (▾): MUC7 (estimated by glycoprotein staining); (Δ): MUC7 (estimated by Western blotting). Results of one representative sample are shown.
Figure 3.
SDS-PAGE analysis of salivary mucins after incubation at 37°C or at 4°C.
Incubated saliva samples at 37°C or 4°C were analyzed by SDS-PAGE. Resolved samples were stained for proteins (A), glycoproteins (B), or incubated with anti-MUC7 and anti-MUC5B (C) antibodies. A: Protein staining of salivary proteins after incubation at 37°C for 0, 5, 20 or 30 min (lanes 1–4), or kept on ice for 5, 20, 30 or 60 min (lanes 5–8). B: glycoprotein staining of salivary proteins after incubation at 37°C for 0, 5, 20, 30 or 60 min (lanes 1–5), or keeping on ice for 5, 20, 30 or 60 min (lanes 6–9). C: Western blotting analysis using anti-MUC7 and anti-MUC5B antibodies after incubation of saliva at 37°C for 0, 5, 20 or 30 min (lanes 1–4), or keeping on ice for 5, 20, 30 or 60 min (lanes 5–8). Arrows in Panel C indicate the positions of bands corresponding to MUC7 and MUC5B. Results of representative saliva sample are shown. Migration positions of molecular weight standards are shown on the left.
Figure 4.
Degradation of MUC7 analyzed by saliva treated with sialidase.
Saliva treated with or without sialidase at 37°C up to 3 hours was analyzed by SDS-PAGE. Results of representative saliva sample are shown. Molecular weight standards are indicated on the left of the results. A: glycoprotein staining; B: Western blotting using anti-MUC7 antibody. The arrow in Panel B indicates the position of bands corresponding to MUC7. Saliva was incubated at 37°C for 0, 30, 60 or 180 min without sialidase (lanes 1–4 in A and B) and incubated at 37 °C for 5, 30, 60 or 180 min with sialidase (lanes 5–8 in A and B). Graphs show half-life time of MUC7 estimated by glycoprotein staining (C) and by Western blotting (D). Densities of MUC7 bands were analyzed densitometrically, and plotted in a semi-log scale. Linear regression curves and half-life time were calculated. (•): control (saliva without sialidase); (○): saliva with sialidase.
Figure 5.
Degradation of MUC7 after treatment of saliva in the presence or absence of sialidase inhibitor.
Saliva was incubated with a sialidase or a sialidase inhibitor up to 60 min at 37°C, followed by SDS-PAGE. Results were shown for a representative saliva sample. Migration positions of molecular weight standards are shown on the left. A: glycoprotein staining. B: Western blotting using anti-MUC7 antibody. An arrow in Panel B indicates the position of bands corresponding to MUC7. Saliva was incubated at 37°C as a controlfor 0 and 60 min (lanes 1–2 in A); for 0, 30, and 60 min (lanes 1–3 in B), incubated at 37°C for 60 min with sialidase (lane 3 in A and lane 4 in B); incubated at 37°C for 60 min with 500 µM sialidase inhibitor (lane 4 in A and lane 5 in B); or incubated at 37°C for 60 min with 5 mM sialidase inhibitor (lane 5 in A and lane 6 in B).
Figure 6.
SDS-PAGE analysis of MUC7 after incubation with protease inhibitors.
The effects of protease inhibitors on the degradation of MUC7 were determined by Western blotting. Saliva was incubated at 37°C for 0, 30, 60 or 90 min with or without protease inhibitors. Results of a representative saliva sample are shown. Migration positions of molecular weight standards are shown on the left. A: MUC7 was detected by Western blotting. An arrow indicates the position of bands corresponding to MUC7. Lanes 1–4: control (saliva without protease inhibitors); lanes 5–8: saliva with a cocktail of protease inhibitors (i. e., PMSF, BZA, NEM and EDTA); lanes 9–12: saliva with EDTA; lanes 13–16: saliva with NEM. B: A graph shows disappearance of MUC7 in a presence of protease inhibitors. The results of Western blotting were analyzed densitometrically, and plotted in a semi-log scale. Linear regression curves were calculated. (•): control (saliva without protease inhibitors); (○): saliva with a cocktail of protease inhibitors (i.e., PMSF, BZA, NEM and EDTA); (▾): saliva with EDTA; (Δ): saliva with NEM. C: MUC7 was detected by Western blotting. An arrow indicates the position of bands corresponding to MUC7. Lanes 1–4: control (saliva without protease inhibitors); lanes 5–8: saliva with a cocktail of protease inhibitors (i.e., PMSF, BZA, NEM and EDTA) as in lanes 5–8; lanes 9–12: saliva with PMSF and BZA; lanes 13–16: saliva with aprotinin, leupeptin, and pepstatin. D: A graph shows dissapearance of MUC7 in the presence of protease inhibitors. Calculation was done as in figure 2B. (•): control (saliva without protease inhibitors); (○): saliva with a cocktail of protease inhibitors (PMSF, BZA, NEM and EDTA); (▾): saliva with PMSF and BZA; (Δ): saliva with aprotinin, leupeptin, and pepstatin.
Table 1.
Classification of protease inhibitors used.