EXPRESSION, STRUCTURE AND FUNCTION OF THE CORNIFIED CELL ENVELOPE

层状鳞状上皮屏障功能的主要组成部分是角化细胞包膜（CE）。这是一种多组分的15纳米厚的高度不溶性蛋白质层，在细胞终末分化时沉积在质膜的内表面。在表皮的情况下，一层5纳米厚的神经酰胺脂质（脂质包膜）附着在外表面。蛋白包膜的不溶性在很大程度上是由于转谷氨酰胺酶使几种结构蛋白交联。研究蛋白质和脂质组分的生物学和组装是本实验室的主要工作。具体而言，我们正在研究：(i)从各种来源分离的ce中蛋白质的交联，以探索哪些蛋白质通过哪些谷氨酰胺和赖氨酸交联在一起，并提供有关结构和功能的信息；（ii）两个关键结构蛋白及其基因，loricrin和小脯氨酸富蛋白（SPR）家族；（iii）与CE共价结合的神经酰胺脂质；（iv）利用免疫金电镜对培养的角质形成细胞中产生的CE片段观察CE组装的最早阶段；(v)尝试使用体外合成脂质囊泡（slv）模型系统重建ce样结构。我们研究了从人包皮表皮角质层、未成熟终分化包皮表皮、培养的诱导终分化的人表皮角质形成细胞、小鼠前胃上皮和人牙龈上皮中分离的CE的特征。我们使用控制蛋白水解来分离ce，分离交联肽，并进行蛋白质测序。在除牙龈外的所有情况下，大部分的蛋白包膜由loricrin（70-80%）和少量的SPRs（2-20%）混合组成。牙龈ce含有bbb50 %的SPRs。在所有情况下，SPRs似乎作为混杂的交叉桥接蛋白发挥作用，通过仅在其头部和尾部结构域上的多个相邻谷氨酰胺和赖氨酸残基将各种蛋白质连接在一起，最常见的是loricrin或它们自己。此外，我们还发现，ce中存在的SPRs的数量与假定的物理特征和暴露于物理上皮创伤之间存在直接相关性：人类或小鼠躯干ce中含有很少或没有SPRs；人包皮表皮ces5 % SPRs；小鼠足垫和唇表皮ce为10%；小鼠前胃CEs 20% SPRs；牙龈ce bb0 50% SPRs。这些数据表明，交叉桥接SPRs有助于调节其表达的ce的生物力学特性。此外，我们已经证明CE与角蛋白中间丝细胞骨架交联，这进一步表明SPRs可能在整个上皮的生物力学特性和需求方面发挥重要作用。此外，我们还发现了许多涉及角蛋白、天青蛋白和其他细胞连接蛋白（包括desmoplakin和envoplakin）的交联。这些发现促使人们对CE组装的最初阶段进行更详细的研究。我们从培养2、3、5和7天的正常人表皮角质形成细胞中回收ce，并将其用于免疫金电镜，并对3和7天的ce进行测序。我们的数据与CE组装沿着质膜在桥粒间位点开始的可能性是一致的，这是通过天青蛋白与自身的头对头和头对尾交联，也可能是与包膜蛋白和外包膜蛋白的交联。不久之后，总苞叶蛋白沉积扩散到桥粒位点，因此沿细胞外围形成了连续的总苞叶蛋白层、包膜蛋白层和周膜蛋白层：这一层可能形成了CE组装后期的支架，包括大量沉积其他蛋白质，如loricrin和SPRs。我们在细菌中表达了人Loricrin，并利用它来表征其结构、生化特性和体外表皮谷氨酰胺转胺酶（TGase）的交联。通过生物物理测量，它在溶液中具有与其多个酪氨酸相关的某些结构。它是一个完整的TGase底物，因为它在体外反应中被所有三种表皮TGase寡聚，尽管具有不同的动力学效率，并且利用不同的谷氨酰胺和赖氨酸。通过比较体外使用的残基和体内使用的残基，我们可以得出结论，TGase 1和TGase 3都是体内氯丙林正确交联所必需的。未来的研究将旨在通过对全长表达蛋白或从其选定部分合成肽的溶液核磁共振方法来确定氯丙林的结构。人类loricrin基因的近端启动子位于转录起始位点上方的第一个160 bp内。c-fos/c-jun蛋白在AP1位点的相互作用对于角化细胞的上皮表达至关重要。与小鼠的loricrin基因不同，钙反应元件位于该区域。我们正在积极地描述位于160 bp区域上游的一系列负面元素。富含脯氨酸的小蛋白质SPRs由三个不同的家族组成，由1到11个成员组成。我们已经分别表达了人类SPR1、SPR2和SPR3蛋白的一个成员，用于体外研究。由于圆二色性，它们在溶液中几乎没有组织结构。存在的结构可归因于中心富含脯氨酸的肽重复序列，并且信号强度与重复次数成正比。在体外交联反应中，SPR蛋白也是三种常见于表皮表达的tgase的完整底物。在研究的所有SPR蛋白中，用于交联的谷氨酰胺和赖氨酸仅位于末端结构域，这表明它们可能具有交联蛋白的功能。然而，细节是不同的。在SPR2蛋白的情况下，TGase 1酶仅在头部结构域使用一个谷氨酰胺残基，在尾部结构域仅使用一个赖氨酸残基进行链间交联，而TGase 3酶使用多个头部和尾部残基进行链间交联。在SPR1蛋白中，我们发现有两个头结构域，称为头A和头b。前者仅由TGase 3酶用于链间交联，而后者仅由TGase 1酶主要用于链内交联。在SPR3蛋白中也发现了类似的情况。此外，我们将这些交联数据与loricrin的交联数据进行了关联，发现TGase 1和TGase 3酶在各自蛋白的共同位点交联。这些数据表明，TGase 3启动loricrin和SPRs的交联，形成小的链间低聚物，然后由TGase 1酶交联到CE上。对SPR2和3蛋白进行了溶液核磁共振结构研究。不幸的是，这些蛋白质在溶液中几乎没有组织结构，并且只获得了短程（2-4）相互作用。然而，数据表明，中心肽重复结构域采用新的ω -环样蛋白折叠。我们利用免疫细胞化学、原位杂交和RT-PCR技术探讨了SPR1和SPR2家族在小鼠上皮细胞中的表达。这两个家族在不同的上皮细胞中表达不同。在SPR1的情况下，表皮的表达量因部位而异，从毛囊间表皮的表达量为零，到脚垫和唇部增厚表皮的表达量非常丰富。在SPR2蛋白的情况下，整个家族在受伤或化学攻击后在表皮中上调。小鼠基因组中的SPR2家族由11个基因组成，这些基因连接在一个约150kbp的簇中，包括一个假定的假基因和一个仅具有头部结构域的过早终止的非常短的蛋白。9种SPR2蛋白之间的差异主要在于中心结构域肽重复的数量，从2.3到11不等，即不同的跨长。下一步的工作将是探索spr作为ce生物力学特性决定因素的可能作用。神经酰胺脂类用甲醇碱性水解的方法可以从包皮中去除共价结合的神经酰胺脂类。通过质谱分析，它们由不同大小的异质分子组成，包括长C28-C34的脂肪醇链和长C18 - C22的鞘氨醇链。附着的神经酰胺的数量对应于CE蛋白质部分的单分子层。这表明，这层神经酰胺对于其他细胞间脂质的组织发挥适当的屏障功能是必不可少的。因此，任何破坏这些神经酰胺或它们与蛋白质CE的有序连接的东西都可能导致鱼鳞病样表型。为了更好地理解这种结构，我们使用了有限的碱性水解，并分离出了几种带有神经酰胺的肽。通过测序，这些肽大部分来源于天卷蛋白的祖先头部结构域序列，以及少量来自其他连接蛋白，包括periplakin， envoplakin和desmoplakin。我们构建了合成脂质囊泡（slv），其组成类似于真核生物的质膜。我们发现，TGase 1酶可以自发地与它们结合。有趣的是，天合蛋白也以钙依赖的方式结合，并且在<1微米时结合，这表明天合蛋白在终末分化开始时表达，就可以在体内附着在质膜上。然而，TGase - 1酶直到Ca浓度升高到100微米时才开始交联天花子蛋白。大多数交联是通过谷氨酰胺496发生的，在其他四个头部结构域残基上也有少量的交联，只有一个赖氨酸残基位于头部结构域，因此天花苷形成头对头和头对尾的低聚物。这些数据对比了bbbb50谷氨酰胺残留物在溶液TGase - 1测定中的利用。此外，我们进行了合成神经酰胺的实验，发现TGase 1酶能够将神经酰胺附着在交联实验中鉴定的相同的四个头结构域谷氨酰胺上，其中三个与在包皮ce上的体内实验中发现的相同。神经酰胺的附着是通过酯的形成发生的。因此，TGase 1酶可以在体内完成CE形成的两个基本方面：(a) CE结构蛋白的交联；(b)神经酰胺层的共价附着。事实上，我们推测由TGase 1失活引起的板层性鱼鳞病的严重表型可能更多地是由于无法附着神经酰胺而不是蛋白质交联。现在需要进一步的工作来探索使用slv系统组装CE的最早阶段。这些研究可能为该病的病因学提供有价值的新见解，并为如何有效治疗鱼鳞病提供线索。

A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the crosslinking of several structural proteins by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the crosslinking of proteins in CEs isolated from a variety of sources to explore which proteins are crosslinked together through which glutamines and lysines, and to provide information on structure and function; (ii) two key structural proteins and their genes, loricrin and the small proline rich protein (SPR) families; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly by use of immunogold electron microscopy on CE fragments produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (slv) model system.CE protein envelope structure and assemblyWe are studying the features of CEs isolated from human foreskin epidermal stratum corneum, immature terminally differentiating foreskin epidermis, cultured human epidermal keratinocytes induced to terminally differentiate, mouse forestomach epithelium, and human gingiva epithelium. We have used controlled proteolysis to dissect apart the CEs, separate crosslinked peptides, and perform protein sequencing. In all cases except the gingiva, the bulk of the protein envelope consists of loricrin (70-80%) admixed with smaller amounts (2-20%) of SPRs. The gingiva CEs contain >50% SPRs. In all cases, the SPRs appear to function as promiscuous crossbridging proteins, by linking together various proteins, most often loricrin or themselves, through multiple adjacent glutamine and lysine residues on their head and tail domains only. In addition, we have found that there is a direct correlation between the amount of SPRs present in CEs and the presumed physical characteristics and exposure to physical trauma of the epithelium: human or mouse trunk CEs contain little or no SPRs; human foreskin epidermal CEs 5% SPRs; mouse foot pad and lip epidermal CEs 10% SPRs; mouse forestomach CEs 20% SPRs; and gingiva CEs >50% SPRs. These data suggest that crossbridging SPRs serve to modulate the biomechanical properties of the CEs in which they are expressed. Moreover, we have shown that the CE is crosslinked to the keratin intermediate filament cytoskeleton, which further suggests that the SPRs may contribute in important ways to the biomechanical properties and requirements of an entire epithelium. In addition, we have recovered many crosslinks involving the keratins, involucrin, and other cell junctional proteins including desmoplakin and envoplakin. These findings prompted a more detailed study on the earliest stages of CE assembly. We recovered CEs from 2, 3, 5 and 7 day cultured normal human epidermal keratinocytes and used them for immunogold electron microscopy, as well as sequencing of the 3 and 7 day CEs. Our data are consistent with the possibility that CE assembly is initiated along the plasma membrane at interdesmosomal sites by head-to-head and head-to-tail crosslinking of involucrin to itself, and perhaps to envoplakin and periplakin. Shortly later, involucrin deposition spreads to desmosomal sites so that a continuous layer of involucrin, envoplakin and perhaps periplakin is formed along the cell periphery: this layer perhaps forms a scaffold for the later stages of CE assembly involving substantial deposition of other proteins such as loricrin and SPRs. Loricrin We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and crosslinking by epidermal transglutaminases (TGase) in vitro. By biophysical measurements it has some structure in solution associated with its multiple tyrosines. It is a complete TGase substrate because it is oligomerized by all three epidermal TGases in in vitro reactions, although with different kinetic efficiencies, and utilization of different glutamines and lysines. From comparisons of the residues used in vitro with those used in vivo from sequencing of CEs, we can conclude that both TGase 1 and TGase 3 are required for the correct crosslinking of loricrin in vivo. Future studies will be aimed at determining the structure of loricrin by use of solution nmr methods on either full length expressed protein, or synthetic peptides from selected portions of it. The proximal promoter of the human loricrin gene resides within the first 160 bp above the transcription initiation start site. Interactions of c-fos/c-jun proteins at an AP1 site are essential for epithelial expression in keratinocytes. Unlike the mouse loricrin gene, a calcium responsive element lies within this region. We are actively characterizing a series of negative elements which lie just upstream of the 160 bp region. Small proline rich proteins SPRs consist of three distinct families consisting of from one to 11 members. We have expressed one member of each of the human SPR1, SPR2 and SPR3 proteins for in vitro studies. By circular dichroism, they have little organized structure in solution. What structure is present can be attributed to the central proline-rich peptide repeats, and the signal strength is proportional to the number of repeats. The SPR proteins are also complete substrates in in vitro crosslinking reactions for the three TGases commonly expressed in the epidermis. In all cases of SPR proteins studied, the glutamines and lysines used for crosslinking are located only on the end domains, suggesting they may function as crossbridging proteins. However, the details are different. In the case of SPR2 proteins, the TGase 1 enzyme uses only one glutamine residue on the head domain, and only one lysine on the tail domain for interchain crosslinking, whereas the TGase 3 enzyme uses multiple head and tail residues for interchain crosslinking. In the SPR1 proteins, we found that there are two head domain regions termed head A and head B. The former are used only by the TGase 3 enzyme for interchain crosslinking, whereas the latter are used only by the TGase 1 enzyme primarily for intrachain crosslinking. A similar situation was found for SPR3 proteins. Moreover, we correlated these crosslinking data with those for loricrin, and we found that the TGase 1 and 3 enzymes crosslink at common sites on the respective proteins. These data suggest that TGase 3 initiates crosslinking of loricrin and SPRs into small interchain oligomers which are later crosslinked to the CE by the TGase 1 enzyme. Solution nmr structural studies on the SPR2 and 3 proteins have been performed. Unfortunately, these proteins have little organized structure in solution and only short range (2-4 ) interactions were obtained. Nevertheless, the data suggest the central peptide repeat domains adopt novel omega-loop-like protein folds. We have explored the expression of the SPR1 and SPR2 families in mouse epithelia by use of immunocytochemistry, in situ hybridization and RT-PCR. Both families are differentially expressed in different epithelia. In the case of SPR1, the amount expressed in the epidermis varies widely with the site, from none in interfollicular epidermis, to very abundant in the thickened epidermis of the foot pad and lip, for example. In the case of the SPR2 proteins, the entire family is upregulated in the epidermis after injury or chemical assault. The SPR2 family in the mouse genome consists of 11 genes linked in a cluster of about 150 kbp, and includes one presumptive pseudogene and one prematurely terminated very short protein possessing only a head domain. The nine SPR2 proteins differ from one another primarily in the numbers of central domain peptide repeats which vary from 2.3 to 11, that is, of different span lengths. Further work will be directed to explore the likely role of the SPRs as determinants of the biomechanical properties of CEs.Ceramide lipids By alkaline hydrolysis in methanol it has been possible to remove covalently bound ceramide lipids from foreskin CEs. By mass spectrometry, these consist of a heterogeneous population of molecules of varying size, containing fatty alcohol chains of C28-C34 long, and sphingosine chains of C18 to C22. The amount of ceramides attached corresponds to a monomolecular layer on the protein portion of the CE. It is suggested that this layer of ceramides is essential for the organization of other intercellular lipids to effect proper barrier function. Thus anything that disrupts these ceramides or their orderly attachment to the protein CE could be predicted to result in an ichthyosis-like phenotype. To better understand this structure, we used limited alkaline hydrolysis, and have isolated several peptides with attached ceramides. By sequencing, most of these peptides derived from the ancestral head domain sequences of involucrin, as well as lesser amounts from other junctional proteins including periplakin, envoplakin and desmoplakin.An in vitro synthetic lipid vesicle system to explore CE assemblyWe have constructed synthetic lipid vesicles (slv) of composition similar to eukaryote plasma membranes. We have found that the TGase 1 enzyme binds spontaneously to them. Interestingly, involucrin also binds in a Ca-dependent manner and at <1 microM, suggesting that involucrin can attach to plasma membranes in vivo as soon as it is expressed during initiation of terminal differentiation. However, the TGase 1 enzyme does not begin to crosslink involucrin until the Ca concentration is raised to >100 microM. Most crosslinking occurs through glutamine 496, with minor amounts at four other head domain residues, and only one lysine residue located on the head domain: thus involucrin forms head-to-head and head-to-tail oligomers. These data contrast the utilization of >50 glutamine residues in solution TGase 1 assays. In addition, we have performed experiments incorporating a synthetic ceramide, and found that the TGase 1 enzyme is capable of attaching the ceramide to the same four head domain glutamines identified in crosslinking experiments, three of which are the same as found in in vivo experiments on foreskin CEs. The ceramide attachment occurs by ester formation. Thus the TGase 1 enzyme can perform two essential aspects of CE formation in vivo: (a) the crosslinking of CE structural proteins; and (b) the covalent attachment of the ceramide layer. Indeed, we speculate that the severe phenotype of the disease lamellar ichthyosis caused by inactivated TGase 1 enzyme may be due more to the inability to attach the ceramide than protein crosslinking. Further work is now needed to explore the earliest stages of CE assembly by use of this slv system. Such studies may provide valuable new insights into disease etiology as well as provide clues as to how to effectively treat ichthyosis disorders.