MS characterization of multiple forms of alpha-amylase in human saliva.
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.
Hirtz
Christophe
C
Laboratory of Physiology, UFR d'Odontologie, Université Montpellier 1, France. christophe.hirtz@univ-montp1.fr
Chevalier
François
F
Centeno
Delphine
D
Rofidal
Valerie
V
Egea
Jean-Christophe
JC
Rossignol
Michel
M
Sommerer
Nicolas
N
Deville de Périère
Dominique
D
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012463:Saliva / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000516:alpha-Amylases / Q000032:analysis* / Q000737:chemistry
D007527:Isoenzymes; D010446:Peptide Fragments; D000516:alpha-Amylases