The structure of the transglutaminase 1 enzyme. Deletion cloning reveals domains that regulate its specific activity and substrate specificity.

Transglutaminase 1 (TGase1) is one of three known enzymes involved in terminal differentiation in stratified squamous epithelia, possibly in the formation of a cornified cell envelope. Because the intact enzyme is particularly difficult to isolate in quantity from keratinocytes for characterization, comparatively little is known about its properties. We have expressed the full-length as well as a series of deletion forms of this enzyme in a bacterial system and analyzed their enzymatic properties. The specific activity of the full-length enzyme isolated and purified from the bacterial lysate was comparable to that of the native enzyme of keratinocytes. Analysis of several deletion constructs demonstrated that removal of the first 60-109 residues, which include sequences involved in membrane association, results in upwards of a 10-fold increase in the specific activity. Deletions beyond residue 109, into sequences conserved within the TGase family of proteins, result in loss of activity. Similarly, as many as 240 residues can be removed from its carboxyl-terminal end before activity is lost. Thus, a molecule of 466 residues, containing virtually only the conserved core sequences of TGases, retains a specific activity comparable to the intact enzyme. In addition, the various deletion forms display wide variations in substrate specificity toward a series of synthetic peptide substrates, designed from possible target TGase1 substrate proteins of epithelia. The data show that sequences between residues 62 and 92 are important in defining the substrate specificity of the TGase1 enzyme system. Furthermore, it may now be possible to design an enzyme with a defined substrate specificity. Together, these data suggest TGase1 has recruited additional sequences on its amino terminus in relation to other members of the TGase family, which have the net effect of permitting sequestration onto membranes, changing its specific activity and modifying its likely substrate specificities.