Proteomic analysis of mice salivary proteins tightly adsorbed on hydroxyapatite

Objectives:

We have already reported that the caries susceptibility of BALB/cA mice was higher than that of C3H/HeN. In addition, those salivary glycoproteins tightly adhered on hydroxyapatite (HAP) in two inbred mouse strains differed not only in the mobility in SDS-PAGE but also in the pH buffering activities.

We carried out the proteomic analysis to clarify the differences of these salivary glycoproteins.

Method:

The whole-saliva was colleted from BALB/cA and C3H/HeN mice by stimulating with pilocarpin sulfate and isoproterenol hydrochloride and was pooled.

HAP-HPLC: The whole-saliva sample was fractionated by HAP-HPLC step-wisely with 50, 70, 100, 150, 200 and 400mM of potassium phosphate buffer.

Differential two-dimensional electrophoresis (2D-PAGE):

HAP-high affinity fractions of the salivary proteins in both mouse strains eluted in 200mM and 400mM in HAP-HPLC were chemically labeled by Cy5 and Cy3 fluorescence and separated by 2D-PAGE in equivalent gel.

Preparation of antibody: Polyclonal rabbit antibody against a main spot obtained from the eluted fraction in 2D-PAGE was prepared.

Glycosilation modification analysis: The eluted fraction was treated with de-glycosilation enzymes of PNGaseF, Neuraminidase and O-Glycosidase. After that the western-blotting analysis was performed with polyclonal antibody.

Results:

In the eluted fractions of 200mM and the 400mM in SDS-PAGE, 45KDa protein was identified in both mice, however the molecular weight of C3H/HeN was slightly higher than BALB/cA.

In differential 2D-PAGE, fluorescent major spots of both mice were piled up, but the molecular weight and PI of C3H/HeN were slightly higher than BALB/cA.

As a result of de-glycosilation and western-blotting, the protein shifted to 30KDa from the 45KDa. The shifted proteins of both mice were the same mobility.

Conclusion:

The salivary proteins of 2 inbred mouse strains strongly adsorbed on HAP were differentially modified by glycosilation. This is suggesting that the difference of pH buffering activity was caused by glycosilation modification.