The Differential Effects of R580A Mutation on Transamidation and GTP Binding Activity of Rat and Human Type 2 Transglutaminase.
Type 2 transglutaminase (TG2) is an acyltransferase, which also undergoes a GTP-binding/GTPase cycle, with guanine nucleotide and calcium binding reciprocally regulating its transamidation (TG) activity. TG2 is expressed ubiquitously throughout the human body and is the predominant neuronal transglutaminase. Given a postulated role for TG2 in a number of physiological and pathological processes including neurodegenerative diseases, it is of critical importance to understand how TG2 and its enzymatic activities are regulated in the cells. The various aspects of TG2 regulation are addressed by using rat and human TG2 proteins, however, despite their homologous structure, regulation of their enzymatic activities may differ, especially in the cellular context. Here, we evaluate the role of Arg580 in human TG2 and Arg579 in rat TG2 in modulating GTP binding and TG activities in vitro and in situ. We confirm the importance of Arg580 and Arg579 in TG2 for GTP binding as their mutation to Ala completely abolished GTP binding activity in both human (R580A) and rat TG2 (R579A). Next, we showed that in transfected human embryonic kidney (HEK) 293 cells, basal in situ TG activity of human R580A TG2 and rat R579A TG2 was significantly greater than their wild-type (WT) counterparts. However, TG activity of the mutants and WT TG2 became equivalent when the intracellular calcium concentration was maximally increased with maitotoxin. Also, in vitro TG activity assay revealed an intriguing difference between rat and human TG2; at a calcium concentration when their activities were maximum, the protein level of human R580A TG2 was lower than its WT counterpart, whereas rat R579A and WT TG2 protein levels were similar. Taken together, our study underscores an essential role of Arg580 in human TG2 and Arg579 in rat TG2 for their GTP binding ability and also describes for the first time that these amino acid residues differentially influence the TG activity of human or rat TG2 by calcium in vitro and in situ.
Ruan
Qingmin
Q
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham Birmingham, AL, 35294-0017, USA.
Tucholski
Janusz
J
Gundemir
Soner
S
Johnson Voll
Gail V W
GV