MS characterization of multiple forms of alpha-amylase in human saliva

Abstract

Alpha-amylase is a major and well-characterized component of human saliva. Recent proteomic studies suggested that this protein could be observed in more than twenty spots on 2-D gels of salivary proteins. The aim of this work was to investigate this unexpected redundancy. 2-D gel electrophoresis was combined with systematic MALDI-TOF MS analysis. More than 140 protein spots identifying the alpha-amylase were shown to constitute a stable but very complex pattern. Careful analysis of mass spectra and simultaneous hierarchical clustering of the observed peptides and of the electrophoretic features of spots allowed one to define three major groups. A main class grouping 90 spots was shown to correspond to full length alpha-amylases that can be assumed to include isoforms and post-translationally modified forms, a subset of this class being demonstrated to be N-glycosylated. A second group included short alpha-amylases that are differently truncated in a non-random manner, very likely in the oral cavity. The last class grouped alpha-amylase forms showing both the N- and C-terminal sequences of the enzyme but displaying a molecular weight that was up to 50% lower than that of the native protein. It is speculated that the last group of alpha-amylase spots could correspond to proteins submitted to internal deletions prior to the secretion.

Changes in the salivary protein profile of morbidly obese women either previously subjected to bariatric surgery or not

“…Increases in salivary amylase spots with apparent molecular masses smaller than the native salivary amylase molecular mass (57 kDa) were observed in the two groups of obese individuals. Low molecular mass salivary amylase isoforms have previously been reported, although their biological role is yet to be investigated [19]. Consequently, a better knowledge of each isoform and its involvement in the development of obesity and/or weight loss may be useful.…”

Human body fluid proteome analysis

“…A significant number of spots on a typical saliva 2-D gel actually correspond to amylases, cystatins, and Igs [369,370]. Therefore, depletion of amylase and Igs prior to 2-DE analysis should improve mapping and identification of saliva proteins.…”

“…Numerous studies have been performed to identify saliva proteins using 2-DE/MS [362][363][364][365][366][367][368][369][370]. A significant number of spots on a typical saliva 2-D gel actually correspond to amylases, cystatins, and Igs [369,370].…”

The Use of Electrophoresis for the Study of Saliva Involvement in Ingestive Behavior

“…However, proteome by its owns does not represent, necessarily, the functionality of these proteins. The complementarity between enzymatic activity data acquisition and electrophoretic protein profiles is also valuable for enzymes present in several different isoforms, being α-amylase and CA6 two examples [83]. In most of the cases the different isoforms are not all involved in the same physiological pathways and frequently express differences in activity and regulation motifs.…”

“…Many salivary proteins present different isoforms [e.g. [83]], with only some of them potentially related to taste sensitivity, making this individual isoform comparison essential.…”