Complication of Type 1 Diabetes in Craniofacial and Dental Hard Tissue

Diabetes mellitus (DM) is a chronic systemic disease arisen under the conditions when the body cannot produce enough insulin or cannot use it effectively. Type 1 diabetes is caused by an autoimmune reaction, where the body’s defense system attacks the insulin-producing β-cells in the pancreas. Type 1 diabetes incidence has been rising all over the world, especially under the age of 15 years. There are strong premonitions of geographic difference; however, the overall annual increase in a number of affected population is estimated to be approximately 3%

Degradation of MUC7 and MUC5B in human saliva.

BACKGROUND: Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis. METHODOLOGY/PRINCIPAL FINDINGS: Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours. CONCLUSIONS/SIGNIFICANCE: MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication

Degradation of MUC7 analyzed by saliva treated with sialidase.

<p>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.</p

Analysis of salivary protein degradation.

<p>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.</p

SDS-PAGE analysis of MUC7 after incubation with protease inhibitors.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069059#pone-0069059-g002" target="_blank">figure 2B</a>. (•): 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.</p

Degradation of MUC7 after treatment of saliva in the presence or absence of sialidase inhibitor.

<p>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).</p

Classification of protease inhibitors used.

<p>Classification of protease inhibitors used.</p

Relationship between Oral Malodor and Glycosylated Salivary Proteins

Volatile sulfur compounds (VSCs), which are major sources of oral malodor, are produced by putrefactive activities of bacteria. Saliva provides easily degradable protein substrates, and most proteins are glycosylated. We hypothesized that oral malodor would be associated with enhanced proteolysis or deglycosylation in saliva. The purpose of this study was to evaluate properties of glycoproteins in saliva and assess their association with VSC levels. Subjects were 88 patients who visited “the Fresh Breath Clinic”, Dental Hospital, Tokyo Medical and Dental University. They were classified into malodor (n = 67) and non-malodor (n = 21) groups. After collecting saliva, the amounts of the total proteins and carbohydrate were determined. Molecular size distributions of salivary proteins/glycoproteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of the total salivary proteins was significantly higher in the malodor group. Major proteins/glycoproteins observed in SDS-PAGE analyses showed similar distributions between the two groups. In the malodor group, the salivary protein concentrations were positively correlated with the CH3SH levels (p < 0.05), and the carbohydrate contents were negatively correlated with the H2S levels (p < 0.05). These results indicated the possibility that salivary proteins/glycoproteins might be related to the malodor generation