Immature cells of sprout apices of Helianthus tuberosus express different forms of transglutaminases homologous to human transglutaminases

Immature cells of sprout apices of Helianthus tuberosus express different forms of transglutaminases homologous to human transglutaminases Simone Beninati1, Rosa Anna Iorio2, Gianluca Tasco2, Donatella Serafini-Fracassini2, Rita Casadio2 and Stefano Del Duca2 1Department of Biology, II University of Roma "Tor Vergata",via della Ricerca Scientifica, 00173, Roma, Italy. 2Department of Biology E. S., University of Bologna, via Irnerio 42, 40126, Bologna, Italy. e-mail: stefano.delduca@unibo.it Immature cells of etiolated apices growing from germinating sprouts of tubers of Helianthus tuberosus (H. t.) showed Ca2+-dependent TGase (TGase, EC 2.3.2.13) activity with fibronectin as substrate, dimethylcasein being substrate only at high Ca2+-concentrations. In three fractions of the 100,000g supernatant of extracted apices eluted at increased NaCl concentrations through a DEAE-cellulose column, three main transglutaminase bands of around 85, 75 and 58 kDa apparent molecular weight were immuno-identified by anti-TGase K type I and rat prostate gland TGase 4. These fractions catalysed the polyamine conjugation at the carboxamide group of N-benzyloxycarbonyl-L-leucine-bound -glutaminyl-residues. The amino acid composition of these TGase protein bands were compared to those of several sequenced TGases of different origin. The composition of the H.t. enzyme with a molecular range of 85 kDa showed a difference of about 2.5% with the average composition of four mammalian TGases 2 and a 3D model was built adopting TGase 2 as template. The H. t. band of 75 kDa showed a difference of about 0.3% with the average composition of three mammalian inactive EPB42, whereas this plant enzyme is active as demonstrated by the formation of glutamyl-derivatives when incubated with lebelled PAs. The 58 kDa form shared a low similarity with the human TGases 2, included their proteolytic fragments, and those of the Arabidopsis recombinant one (24-28 % difference), and even more with that of Streptoverticillium (41 % difference) and of the two maize sequences of similar molecular mass (51-52 % difference), which however are very dissimilar from all other known TGases. By comparison to known plant TGases, these novel TGases are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells of the sprout apices. These data also confirm that in plants there are multiple active forms of TGases in the same organ and that plant and animal enzymes probably are similar also structurally and not only, as already known, for their catalytic activity.