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e-NH2之间形成交联。这种交联是一种在真核生物中不能被切割的异肽键。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。它们与多种结构蛋白交联形成凝固的细胞包膜，这是上皮屏障功能的主要组成部分。我们正在详细研究每一种酶。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。为了解决这个问题，我们已经开发了在杆状病毒系统中表达它的方法，在这种系统中，它的合成修饰基本上与在角质形成细胞中相同，但由于它没有被蛋白水解，因此更稳定。通过这种方式，我们能够产生微量的酶，这些酶可以无限期地储存起来。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。我们已经在杆状病毒中表达了一些已知的突变。大多数产生的产物没有或只有非常低的活性。然而，另外两种产物的活性是野生型酶的10倍。使用相关因子XIIIa TGase酶的可用结构坐标检查它们的结构，我们预计这两个突变导致异常稳定的蛋白质。为了验证这一想法，我们接下来在羧基端用附着的组氨酸标签表达这些相同的形式，并将它们用于转染培养的角质形成细胞的实验。通过his-tag抗体的特异性免疫沉淀，我们发现这些突变蛋白在体内没有被蛋白水解加工成高活性的形式。这些数据表明，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的合成修饰而不能被细胞利用。正在进行的工作旨在了解TGase 1酶锚定在膜上的机制，以及这如何影响其对某些已知底物的底物特异性，包括loricrin，富含脯氨酸的小蛋白，involucrin和plakin家族的各种成员，包括envoplakin和desmoplakin。此外，现有数据表明，90残基膜锚定段控制TGase 1的活性。我们将尝试在细菌/杆状病毒系统中表达该片段，以研究其结构和功能特性。转谷氨酰胺酶2我们对这种酶的主要研究重点是通过晶体的x射线衍射获得原子分辨率的结构信息。迄今为止，我们已经开发出大规模制备杆状病毒酶的方法。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。tgm3基因的近端启动子区位于转录起始位点上方的前126 bp，由Sp1基序组成，由邻近的et -like基序调节。这些足以赋予上皮特异性表达。这个区域也含有钙反应元素。此外，我们已经发现单链DNA结合蛋白可能作为负控制元件的证据。对该启动子的研究将继续进行。杆状病毒中已大量表达可活化的前tgase 3酶。初步的工作已经证明了小晶体的形成，我们利用布鲁克海文同步x射线设备在3.5 A水平上产生了结构信息。我们现在正在开发更大晶体生长的程序，这可能适合于完整的结构确定。我们发现在杆状病毒合成过程中用硒代蛋氨酸标记可以增强x射线信号和分辨率。这些研究将继续进行。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。这些酶的表达已被特异性抗体的免疫沉淀反应和间接免疫荧光法证实。此外，tgase 1和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b-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。最后，我们发现当成纤维细胞受到低水平的试剂如SDS的刺激时，它们会大大提高TGase 1酶的表达，该酶开始交联各种细胞骨架蛋白，包括vimentin和actin。初步数据表明，这不会促进细胞凋亡。然而，该系统可以作为接触性皮炎的实验模型。

Transglutaminases (TGases) catalyze the formation of a crosslink between a donor amide group of a protein-bound glutamine residue and an acceptor e-NH2 of a protein-bound lysine residue. This crosslink is an isopeptide bond that cannot be cleaved in eukaryote 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 crosslink a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying each of these enzymes in detail. 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 this problem, we have developed methods for its expression in the baculovirus system in which it is postsynthetically modified essentially the same ways as in keratinocytes, but as it is not proteolyzed it is more stable. In this way, we are able to produce microg amounts of enzyme which can be stored for use 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 some of the known mutations. Most result in a product of no or only very low activity. However, two others result in a product of >10-fold activity than the wildtype enzyme. Examination of their structures using the coordinates of the available structure of the related factor XIIIa TGase enzyme, we anticipate that these two mutations result in an unusually stabilized protein. In order to test this idea, we next expressed these same forms with an attached histidine tag at the carboxy terminus, and used them for transfection experiments into cultured keratinocytes. By specific immuno-precipitations with the his-tag antibody, we found that these mutant proteins were not proteolytically processed into highly active forms in vivo. These data 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. Ongoing work is directed toward an understanding of the mechanism by which the TGase 1 enzyme is anchored to membranes and how this affects its substrate specificity toward certain known substrates including loricrin, small proline rich proteins, involucrin and various members of the plakin family including envoplakin and desmoplakin. In addition, available data suggest the 90 residue membrane anchorage segment controls TGase 1 activity. We will attempt to express this fragment in bacteria/baculovirus systems in order to study its structure and functional properties. Transglutaminase 2Our 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 enzyme in baculovirus. Ongoing work will attempt to purify active forms of the enzyme in order to initiate crystallization trials. Transglutaminase 3The 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 crosslinking in vivo of several important substrates involved in barrier or other functions, including loricrin, small proline rich proteins, and trichohyalin. The proximal promoter region of the TGM 3 gene is located within the first 126 bp above the transcription start site, and consists of an Sp1 motif modulated by adjacent ets-like motifs. These are sufficient to confer epithelial-specific expression. This region also contains a calcium responsive element. In addition, we have found evidence for a single-stranded DNA binding protein that may serve as a negative controlling element. Studies on this promoter will continue. Activatable pro-TGase 3 enzyme has been expressed in large quantities in baculovirus. Preliminary work has demonstrated the formation of small crystals, from which we have generated structural information at the 3.5 A level using the Brookhaven synchronton X-ray facility. We are now developing procedures for the growth of larger crystals that may be suitable for complete structural ascertainment. We have found that labeling during synthesis in baculovirus with selenomethionine may enhance the X-ray signals and resolution. These studies will continue. Expression of TGases in non-epithelial tissues: roles in degenerative diseasesBy RT-PCR methods, we have found that TGases 1 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 reactions with specific antibodies, and by indirect immunofluorescence methods. Moreover, mRNA and enzyme levels of TGases 1 and 3 are upregulated 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 crosslink 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 b-amyloid protein crosslinked 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 crosslinked 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 anticipate that such studies may provide valuable new insights into both disease etiology as well as methods to control the degenerative diseases processes. Finally, we have found that when fibroblasts are irritated by low levels of a reagent such as SDS, they greatly elevate the expression of the TGase 1 enzyme, which begins to crosslink various cytoskeletal proteins including vimentin and actin. Preliminary data suggest this does not promote apoptosis. However, this system may serve as an experimental model of contact dermatitis.