Characterization of recombinant transglutaminases 1 and 3 expressed in baculovirus system

摘要

Transglutaminase (TGase) is a calcium ion-dependent enzyme that catalyze the formation of isopeptide cross-links in proteins between the gamma -carboxamide groups of glutamme residues and epsilon -amino groups of lysine residues. Both TGases of keratinocyte-type (TGase 1) and epidermal-type (TGase 3) are involved in the formation of the cornified cell envelop, which serve a vital barrier function for the skin, by cross-linking of a variety of structural proteins in the epidermis. TGase 1 and TGase3 are 107 kDa and 77kDa protein, respectively. Both enzymes are limitedly proteolysed during epidermal differentiation. It has been difficult to isolate sufficient quantities of native enzymes from tissues or to obtain recombinant proteins in a bacterialexpression system for biochemical studies on its properties of the TGases. Therefore, we circumvented these problems by expressing recombinant full-length cDNA of the TGases in baculovirus-infected insect cells. The expressed proteins were purified to homogeneity by successive chromatography and HPLC. Calpain, that had been a candidate for activating enzyme, was not effective for activation of TGase 3 zymogen. In the case of TGase 1, the calpain-proteolysed form was highly sensitive to the calcium-ionfor TGase activity.