Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase.
Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross-links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine-containing peptide was used as probe for labeling the amino-donor sites. SDS gel electrophoresis of a time-course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino-donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino-donor residues modified by tissue transglutaminase.
Orru
Stefania
S
Dipartimento di Chimica, Università di Salerno, Baronissi, Salerno, Italy.
Ruoppolo
Margherita
M
Francese
Simona
S
Vitagliano
Luigi
L
Marino
Gennaro
G
Esposito
Carla
C
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry*
D010455:Peptides; D005973:Glutamine; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011503:Transglutaminases; D008239:Lysine