HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivary statherin generated by transglutaminase 2.
In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (M(av)5380.0 +/- 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 +/- 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique Ione-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (> 95%), with the percentage of Gln-39 implicated in the cross-linkiing being less than 5%. The main derivative was named cyclostatherin Q37. Guineapig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 +/- 0.06 microM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".
Cabras
Tiziana
T
Department of Sciences Applied to Biosystems, Cagliari University, Monserrato Campus, Monserrato, CA, Italy.
Inzitari
Rosanna
R
Fanali
Chiara
C
Scarano
Emanuele
E
Patamia
Maria
M
Sanna
Maria T
MT
Pisano
Elisabetta
E
Giardina
Bruno
B
Castagnola
Massimo
M
Messana
Irene
I
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D019204:GTP-Binding Proteins / Q000502:physiology*; D006168:Guinea Pigs; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012463:Saliva / Q000201:enzymology*; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011503:Transglutaminases / Q000502:physiology*
C018149:STATH protein, human; D012471:Salivary Proteins and Peptides; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins