title: Top-down HPLC-ESI-MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine-rich proteins and potential substrate of transglutaminase.
abstract: During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.
authors: Dipartimento di Scienze della Vita e dell'Ambiente, Univ. di Cagliari, Cagliari, Italy.
authors: Tiziana
authors: T
authors: Cabras
authors: Federica
authors: F
authors: Iavarone
authors: Davide
authors: D
authors: Pirolli
authors: Maria Cristina
authors: MC
authors: De Rosa
authors: Alberto
authors: A
authors: Vitali
authors: Gavino
authors: G
authors: Faa
authors: Massimo
authors: M
authors: Cordaro
authors: Irene
authors: I
authors: Messana
authors: Jörgen
authors: J
authors: Ekström
authors: Massimo
authors: M
authors: Castagnola
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005973:Glutamine / Q000737:chemistry*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051381:Rats; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013363:Submandibular Gland / Q000737:chemistry*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D012471:Salivary Proteins and Peptides; C000717609:Tgm2 protein, rat; D005973:Glutamine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases
keywords: Glutamine-rich protein; Rat; Saliva; Submandibular; Top-down