Epidermal Transglutaminases

转谷氨酰胺酶（TGases）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ε-NH2之间形成交联。这种交联是在脊椎动物生物体中不能被切割的异肽键。因此，最终结果是形成永久的、稳定的、不溶性的大分子蛋白质复合物。在表皮和其他复层鳞状上皮中，表达九种已知的TGase酶中的几种。特别地，TGases 1、2和3交联多种确定的结构蛋白以形成皮质细胞包膜，其是上皮屏障功能的主要组分。我们正在详细研究这些酶中的每一种，以及它们在疾病中的作用。 转氨酶1 培养的角质形成细胞或包皮表皮细胞中的TGase 1酶是复杂的，因为它以多种可溶性和膜结合全长以及蛋白水解加工的形式存在。大多数酶通过肉豆蔻酸酯与膜结合，并锚定在TGase 1酶特有的氨基末端片段上。各种形式的比活性显示出广泛的变化，但这些是难以测量的，因为酶本身不稳定，容易被蛋白水解降解。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。本实验室以前的工作表明，编码TGase 1酶的TGM1基因突变导致常染色体隐性遗传疾病板层状鱼鳞病。我们已经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。这些研究与基于与TGase 3和因子XIIIa酶的比较结构的结构分析相结合。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。这些研究提供了了解其结构和底物特异性的机会。转氨酶2 我们对这种酶的主要关注点是通过晶体的X射线衍射获得原子分辨率的结构信息。迄今为止，我们已经开发了杆状病毒中活性酶的大规模制备和纯化方法。正在进行的工作将包括结晶试验。 转氨酶3 TGase 3酶在许多上皮细胞类型中表达，最初作为无活性的酶原，需要通过特异性切割进行蛋白水解活化。此外，来自该实验室的数据表明，它是参与屏障或其他功能的几种重要底物（包括兜甲蛋白、富含脯氨酸的小蛋白和透明质酸）体内交联的优选酶。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50 kDa活性形式。每一种都已结晶，其结构已通过X-射线衍射方法解析至2.5埃或更小。例如，活化形式获得了活性所需的3个钙离子，与酶原相比，这导致结构的显著变化。这些数据为TGase酶反应机理的新认识提供了基础。在活性位点区域或其附近已经鉴定出几个残基，这些残基可能赋予酶底物特异性。这些研究将继续进行。希望他们能让我们合理地设计这种酶的特异性抑制剂。 TGases在非上皮组织中的表达及其在退行性疾病中的作用 通过RT-PCR方法，我们已经发现TGases 1、2和3在各种非上皮组织中广泛表达，特别是包括脑内的各种组织、结缔组织、成纤维细胞和肌肉。因此，认为这些组织仅表达胞质TGase 2酶。这些酶的表达已被证实通过免疫沉淀和间接免疫荧光方法，使用特异性探针。此外，TG酶1和3的mRNA和酶水平在病理状况中上调，包括阿尔茨海默病（AD）、散发性包涵体肌炎（SIBM）和乳糜泻。总之，这些数据表明，TG酶1和/或3的水平升高与疾病发病机制相关，并直接促成不溶性交联体的形成，所述交联体干扰正常细胞功能并因此导致退行性疾病。根据足够量组织的可用性，进一步的工作将针对TGase 1和3酶的形式的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、II型糖尿病和亨廷顿病，所有这些疾病都有异常高水平的TGase表达。 TGase家族的新成员 最近发现了三个新的TGase家族成员，其中至少有两个，TGase 5和6，在上皮细胞中大量表达。我们已经获得了这两种表达载体，并将表达这些载体，目的是进行额外的结构研究。此外，我们将探讨它们在疾病中的作用。

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 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. These studies offer an opportunity 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 and purification of the active enzyme in baculovirus. Ongoing work will involve 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. For example, the activated form acquired 3 calcium ions that are required for activity and which cause a significant change in structure as compared to the zymogen. These data have provided the basis for a new understanding of the TGase enzyme reaction mechanism. 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 Alzheimers Disease (AD), sporadic inclusion body myositis (SIBM) and celiac disease. 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 Huntingtons Disease, all of which have aberrantly high levels of TGase expression. New members of the TGase family Three new members of the TGase family have been discovered recently, and at least two of these, TGases 5 and 6, are abundantly expressed in epithelia. We have acquired expression vectors for both and will express these with the aim of performing additional structural studies. Further, we will explore their roles in diseases.