Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation.
Tissue transglutaminase (tTG) is a Ca2+-dependent cross-linking enzyme that participates in the apoptotic machinery by irreversibly assembling a protein scaffold that prevents the leakage of intracellular components. In the present study a single-chain antibody fragment (scFv) detecting tTG is described. We demonstrate that TG/F8 scFv, selected from a phase display library of human V-gene segments by binding to guinea-pig liver tTG, can react with human tTG both in Western blot and in immunohistochemistry. The specific detection of tTG by TG/F8 in human thymocytes is verified by mass spectrometric analysis of the purified protein. Furthermore, we demonstrate that in lymphoid cells tTG is cleaved by caspase 3 during the late phase of apoptotic death, concomitant to DNA fragmentation, and that such cleavage causes loss of cross-linking function. We propose tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.
Fabbi
M
M
Istituto Nazionale per la Ricerca sul Cancro, L.go R.Benzi 10, 16132 Genova, Italy. fabbi@ermes.cba.unige.it
Marimpietri
D
D
Martini
S
S
Brancolini
C
C
Amoresano
A
A
Scaloni
A
A
Bargellesi
A
A
Cosulich
E
E
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers / Q000737:chemistry; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007942:Leukemia, Experimental / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000166:cytology; D051379:Mice; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013950:Thymus Gland / Q000737:chemistry / Q000166:cytology; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*
D015415:Biomarkers; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D019204:GTP-Binding Proteins