Epidermal Transglutaminases

转谷氨酰胺酶(TGase)催化蛋白质结合的谷氨酰胺残基的供体酰胺基团和蛋白质结合的赖氨酸残基的受体epsilon-NH2之间形成交联键。这种交联键是一种在脊椎动物中不能裂解的异肽键。因此，最终结果是形成一种永久的、稳定的、不溶于水的大分子蛋白质复合体。在表皮和其他复层鳞状上皮中，九种已知的TGase酶中的几种都有表达。特别是，TGase1、2和3使多种已定义的结构蛋白交联，形成角化的细胞膜，这是上皮屏障功能的主要组成部分。我们正在详细研究这些酶，以及它们在疾病中的作用。 转谷氨酰胺酶1是培养的角质形成细胞或包皮表皮细胞中的转谷氨酰胺酶1，是一种复杂的酶，因为它以多种形式存在，既有可溶性的，也有膜结合的，也有蛋白水解性的。该酶大部分是以肉豆蔻酸酯和氨基末端锚定的方式结合在膜上，这是TGase 1酶所特有的。各种形式的酶在特定活性上表现出很大的差异，但这些变化很难测量，因为酶天生不稳定，很容易被蛋白质分解降解。为了解决结构和功能问题，我们已经成功地在杆状病毒系统中表达。该实验室以前的工作表明，编码TGase 1酶的TGM1基因突变会导致常染色体隐性遗传性板层鱼鳞病。我们在一个日本家庭中研究了它的分子基础，并报告了两个新的TGM1突变(R348X和Y365D)。分子对接研究表明，该先证者的LI表型可以通过预测母系和父系衍生的每个突变都会导致酶活性丧失来解释。这项研究发表了。在另一项这类研究中，我们发现了两个表皮TGM1活性显著降低的自愈性火棉婴儿兄弟姐妹，我们发现了TGM1复合杂合突变G278R和D490G。在高压下对突变蛋白质的分子模拟和生化分析表明，G278R的活性显著降低，D490G的水分子被螯合，从而将突变的酶锁定在子宫内的非活性反式构象中。出生后，这些水分子被去除，这种酶预计会异构化回到部分活跃的顺式结构，这解释了这种皮肤状况的戏剧性改善。这项研究也发表了 我们继续用X射线衍射法对TGase3进行了结构分析。通过这种方式，我们希望更好地了解这种酶在皮肤中的作用。TGase 3在多种上皮细胞中表达，最初是一种不活跃的酶原，需要通过特定的切割来激活蛋白水解酶。为此，我们开发了在杆状病毒系统中大规模表达和纯化几种形式的TGase 3的方法。这些包括酶原、激活酶和50 kDa的活性形式。在过去的一年里，我们重点研究了二价阳离子结合的调控作用。具体地说，我们解决了在钙和/或镁存在下TGM3的三种形式的结构，这为每种钙离子对激活和活性的贡献提供了新的见解。首先，我们发现第一位点的钙离子很难交换，它的结合亲和力为Kd=0.3 mM，这表明它对酶的稳定是重要的。第二位可以被碱土金属第二族中的一些稀土元素占据，但只能被第二族碱土金属中的钙占据，活性需要占据它的位置。第三位点可以被一些稀土元素、钙或镁占据；然而，当有镁存在时，酶就不活跃，通道关闭。因此，两个位点2和3上的钙结合在一起打开了通道。我们推测，通道开放的操作可能受细胞内阳离子水平的控制。总之，这些数据对酶的反应机制有重要的影响：通道的打开可能控制着活性部位底物的访问和操纵。

Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, several of the nine known TGase enzymes are expressed. In particular, TGases 1, 2 and 3 cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. Most of the enzyme is membrane bound by way of myristate and anchorages on the amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have been successful in expression in baculovirus systems. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We examined its molecular basis in a Japanese family and reported two novel TGM1 mutations (R348X, and Y365D). Molecular docking studies revealed that the LI phenotype of this proband can be explained by the prediction that each of the maternally and paternally derived mutations will result in loss of enzyme activity. This study was published. In another investigation of this kind, two self-healing collodion baby siblings with markedly diminished epidermal TGM1 activity we were found to have compound heterozygous TGM1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition. This study was also published We have continued to perform structural analyses of TGase 3 by X-ray diffraction. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. TGase 3 is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. To this end, we developed methods for the large-scale expression and purification of several forms of TGase 3 in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Over the past year, we focussed on regulatory effect of divalent cation binding. Specifically, we solved the structures of three forms of TGM3 in the presence of Ca2+ and/or Mg2+ which provide new insights on the contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty and it has a binding affinity of Kd= 0.3 mM, which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy is required for activity. Site three can be occupied by some lanthanides, Ca2+ or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.