The Selective Adsorption of Salivary Amylase in Human Acquired Enamel Pellicle

Salivary amylase is one of the predominant proteins in saliva. In vitro studies have shown that amylase exhibits affinity for hydroxyapatite (HA). In a recent investigation on the characterization of in vivo formed human acquired enamel pellicle (EP) the presence of amylase could be substantiated. Salivary amylase exists in both glycosylated and non-glycosylated forms and was found to consist of multiple isoforms. However, the role of both forms of amylase and how these isoforms differ still remains largely unknown. Objectives: The goal of the present study was to investigate the distribution pattern of amylase in glandular secretions, whole saliva (WS) and EP. Methods: Salivary parotid secretions (PS), submandibular/sublingual secretions (SMSL), WS and EP were collected from healthy individuals. In vitro pellicle samples (PS-HA and WS-HA) were obtained by incubating PS or WS with HA powder (1 ml: 5 mg) for 2 h at room temperature. Samples containing 100 µg of proteins/peptides were analyzed by two-dimensional gel electrophoresis (1. dimension: iso-electric focusing; 2. dimension: SDS-PAGE). Gels were stained with silver or blotted onto PVDF membranes for immunodetection. Selected samples were analyzed by mass spectrometry (MS). Results: The data showed that the non-glycosylated forms of amylase were predominant in PS, WS as well as in PS-HA and WS-HA but both forms contributed equally to SMSL. Interestingly, glycosylated amylases heavily predominated over non-glycosylated species in EP. At least 8 isoforms could be clearly observed in all samples except EP. Each of the 8 isoforms was found to pair up for both glycosylated and non-glysosylated amylases exhibiting identical pI with molecular weight difference of 3 kDa. MS data showed that these isoforms differ in post-translational modification. Conclusion: EP formation comprises selective adsorption of glycosylated amylases. Structural analyses are in progress to investigate the significance of this finding. Supported by NIH/NIDCR DE05672 DE07652 and DE07206.