Human saliva proteome reveal unexpected forms of alpha-amylase

Alpha-amylase is the major protein of human saliva. Recent proteomic studies on salivary proteins suggested that alpha-amylase could be observed in more than twenty spots on two-dimensional gels, with no clear explanation of this unforeseen phenomenon. Objective: The aim of this work was to investigate the unexpected redundancy of Alpha-amylase on 2D gels of salivary proteins. Methods: Parafilm-stimulated whole saliva was collected 2 hours after usual breakfast time and complemented with a protease inhibitor cocktail. Saliva samples were centrifuged at 10,000 g for 15 min, and the supernatant was frozen at -80°C. Salivary proteins were extracted using Acetone/TCA method, separated using two-dimensional gel electrophoresis, identified by MALDI-TOF mass spectrometry analysis and mass spectra corresponding to alpha-amylase were carefully inspected and compared. Results: More than 140 protein spots of salivary alpha-amylase formed a stable but very complex pattern. Simultaneous hierarchical clustering of the observed peptides allowed us to define three major groups. The main class, grouping 90 spots corresponded to full-length alpha-amylases that can be assumed to include isoforms and post-translationally modified forms. A second group, included short alpha-amylase, was truncated in a non-random manner. The last class grouped alpha-amylase forms with both the N- and C-terminal sequences of the enzyme but displaying a molecular weight up to 50 % lower than the native protein. Conclusion: This work exhibited for the first time the presence of multiple forms of alpha amylase, including native isoforms, degradation forms and unexpected smaller forms. Internal deletions were speculated to occur during maturation of alpha-amylase belonging to such unexpected forms.