Epidermal Transglutaminases

谷氨酰胺转酶（TGases）催化在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。这种交联是一种异肽键，在脊椎动物中不能被切断。因此，最终的结果是形成一个永久的、稳定的、不溶的大分子蛋白质复合物。在表皮和其他层状鳞状上皮中，至少有三种不同的TGase酶，TGase 1、2和3表达。它们交联多种确定的结构蛋白，形成凝固的细胞包膜，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。谷氨酰胺转氨酶1：在培养的角质形成细胞或包皮表皮细胞中，谷氨酰胺转氨酶1是复杂的，因为它以多种可溶性和膜结合的全长形式存在，也以多种蛋白水解形式存在。胞质溶胶和细胞膜之间的分配是由肉豆蔻酸酯和棕榈酸酯对一簇半胱氨酸残基的差异酰化控制的，这些半胱氨酸残基位于TGase 1所特有的膜锚定氨基末端。各种形式的酶在特定的活性上表现出很大的差异，但这些差异很难测量，因为这种酶本身就不稳定，很容易被蛋白质水解降解。为了解决结构和功能问题，我们已经测试了几种可用的方法来大规模表达这种酶，迄今为止还没有成功，尽管我们能够生产微克数量的酶，可以无限期储存。该实验室先前的工作表明，编码TGase 1酶的TGM1基因突变导致常染色体隐性遗传病板层性鱼鳞病。我们在杆状病毒中表达了几种已知的突变，以探索酶活性丧失的基础。这些研究结合了基于与TGase 3和factor XIIIa酶比较结构的结构分析。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。例如，一些突变清楚地表明，板层状鱼鳞病可能是由酶活性不足引起的，或者是一种酶形式没有适当地合成修饰而不能被细胞利用。此外，这些研究提供了迄今为止了解其结构和底物特异性的唯一机会。转谷氨酰胺酶2我们研究这种酶的主要目的是通过晶体的x射线衍射获得原子分辨率的结构信息。迄今为止，我们已经开发了大规模制备杆状病毒活性酶的方法。正在进行的工作将试图纯化酶的活性形式，以便开始结晶试验。谷氨酰胺转酶3在许多上皮细胞类型中表达，最初作为一种无活性的前酶，需要通过特异性裂解激活蛋白水解。此外，本实验室的数据表明，它是几种重要的屏障或其他功能底物在体内交联的首选酶，包括氯丙氨酸、富含脯氨酸的小蛋白质和毛透明蛋白。我们已经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酶的方法。这些包括前酶，活化酶和50kda的活性形式。每一种都已结晶，并用x射线衍射方法分辨出2.5 A或更小的结构。每个细胞都含有2到3个钙离子，这是维持活性所必需的。在活性位点区域或附近发现了几个残基，这些残基可能赋予酶的底物特异性。这些研究将继续进行。希望它们能让我们合理地设计出针对这种酶的特定抑制剂。tgase在非上皮组织中的表达：在退行性疾病中的作用通过RT-PCR方法，我们发现tgase 1、2和3在多种非上皮组织中广泛表达，特别是在大脑、结缔组织、成纤维细胞和肌肉中的各种组织中。在此之前，这些组织被认为只表达胞质TGase 2酶。这些酶的表达已被免疫沉淀法和间接免疫荧光法用特异性探针证实。此外，tgase 1和tgase 3的mRNA和酶水平在病理条件下上调，包括阿尔茨海默病？病（AD）和散发包涵体肌炎（SIBM）。我们发现TGase抗原与两种疾病的包涵体/老年斑共定位。此外，我们从这些AD和SIBM的组织切片中分离出含有大量由TGases形成的异肽交联的不溶性蛋白，表明它们之间存在直接的致病关系。此外，在SIBM的情况下，我们进行了测序分析，以证明存在大量与自身交联的β -淀粉样蛋白、肌球蛋白和desmin肌肉蛋白。总之，这些数据表明，tgase 1和/或3水平升高与疾病发病机制相关，并直接导致不溶性交联体的形成，从而干扰正常细胞功能，从而导致退行性疾病。根据足够数量的组织的可用性，进一步的工作将指向TGase 1和3酶的形式分析和更详细的测序分析。我们正在对其他疾病进行平行研究包括乳糜泻，二型糖尿病和亨廷顿舞蹈症？这些疾病都有异常高水平的TGase表达。此外，最近还发现了TGase家族的三个新成员。这些基因的表达也将在这些疾病状态下进行测试。我们预计这些研究可能为疾病病因学以及控制退行性疾病过程的方法提供有价值的新见解。

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, at least three different TGase enzymes, TGases 1, 2 and 3, are expressed. They 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. The partitioning between the cytosol and membranes is controlled by differential acylation by myristate and palmitate of a cluster of cysteine residues located on a membrane anchorage 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 tested several available methods for the large-scale expression of this enzyme, which to date have not been successful, although we are able to produce microgram amounts of enzyme which can be stored indefinitely. 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 have expressed in baculovirus several of the known mutations to explore the bases of loss of enzyme activity. These studies have been coupled with structural analyses based on comparative structure with the TGase 3 and factor XIIIa enzymes. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. For example, some mutations clearly suggest that lamellar ichthyosis disease may be caused by either insufficient enzyme activity, or an enzyme form that is not appropriately postsynthetically modified and cannot be utilized by the cell. In addition, these studies offer the only opportunity to date to understand its structure and substrate specificity. Transglutaminase 2 Our main focus of this enzyme is to obtain atomic resolution structural information by X-ray diffraction of crystals. To date, we have developed methods for the large scale preparation of the active enzyme in baculovirus. Ongoing work will attempt to purify active forms of the enzyme in order to initiate crystallization trials. Transglutaminase 3 The TGase 3 enzyme is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. In addition, data from this laboratory have shown that it is the preferred enzyme for cross-linking in vivo of several important substrates involved in barrier or other functions, including loricrin, small proline rich proteins, and trichohyalin. We have developed methods for the large-scale expression and purification of several forms of the TGase 3 enzyme in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Each of these has been crystallized and the structures have been resolved by X-ray diffraction methods to 2.5 A or less. Each possess 2 or 3 calcium ions that are required for activity. Several residues have identified at or near the active site region that may confer substrate specificity for the enzyme. These studies will continue. Hopefully, they will allow us to rationally design specific inhibitors for this enzyme. Expression of TGases in non-epithelial tissues: roles in degenerative diseases By RT-PCR methods, we have found that TGases 1, 2 and 3 are widely expressed in a variety of non-epithelial tissues, including in particular, various tissues within the brain, connective tissues, fibroblasts and muscle. Heretofore, these tissues were thought to express only the cytosolic TGase 2 enzyme. The expression of these enzymes has been confirmed by both immunoprecipitation and indirect immunofluorescence methods using specific probes. Moreover, mRNA and enzyme levels of TGases 1 and 3 are up-regulated in pathological conditions, including Alzheimer?s Disease (AD) and sporadic inclusion body myositis (SIBM). We found co-localization of the TGase enzyme antigens with the inclusion bodies/senile plaques of both diseases. In addition, we isolated insoluble proteins from tissue slices from these AD and SIBM which contained substantial amounts of isopeptide cross-link formed by TGases, suggesting a direct causative link. Also, in the case of SIBM, we performed sequencing analyses to demonstrate the presence of significant amounts of beta-amyloid protein cross-linked to itself, myosin and desmin muscle proteins. Together, these data demonstrate that elevated levels of TGases 1 and/or 3 correlate with disease pathogenesis and contribute directly to the formation of insoluble cross-linked bodies that interfere with normal cellular function and thus degenerative disease. Depending on availability of adequate amounts of tissue, further work will be directed toward analysis of the forms of the TGase 1 and 3 enzymes and more detailed sequencing analyses. We are performing parallel studies with other diseases including celiac disease, type II diabetes and Huntington?s Disease, all of which have aberrantly high levels of TGase expression. In addition, three new members of the TGase family have been discovered recently. The expression of these will also be tested in these disease states. We anticipate that such studies may provide valuable new insights into both disease etiology as well as methods to control the degenerative disease processes.