Kim S Y, Kim I G, Chung S I, Steinert P M
Skin Biology Branch, NIAMS, National Institutes of Health, Bethesda, Maryland.
J Biol Chem. 1994 Nov 11;269(45):27979-86.
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.
转谷氨酰胺酶1（TGase1）是已知参与复层鳞状上皮终末分化的三种酶之一，可能与角质化细胞包膜的形成有关。由于完整的该酶特别难以从角质形成细胞中大量分离以进行表征，因此对其特性了解相对较少。我们已在细菌系统中表达了该酶的全长以及一系列缺失形式，并分析了它们的酶学特性。从细菌裂解物中分离纯化得到的全长酶的比活性与角质形成细胞的天然酶相当。对几种缺失构建体的分析表明，去除包括参与膜结合的序列在内的前60 - 109个残基后，比活性会增加10倍以上。缺失超过第109位残基，进入TGase蛋白家族内保守的序列，会导致活性丧失。同样，在其羧基末端去除多达240个残基后才会失去活性。因此，一个仅包含TGases保守核心序列的466个残基的分子，其保留的比活性与完整酶相当。此外，各种缺失形式对一系列由上皮细胞可能的靶标TGase1底物蛋白设计的合成肽底物表现出广泛的底物特异性差异。数据表明，第62至92位残基之间的序列对于定义TGase1酶系统的底物特异性很重要。此外，现在有可能设计出具有确定底物特异性的酶。总之，这些数据表明，与TGase家族的其他成员相比，TGase 在其氨基末端募集了额外的序列，其净效应是允许其与膜结合，改变其比活性并改变其可能的底物特异性。
