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内容記述 |
In multi-cellular organisms, establishment of compositionally destlnct fluid compartments by epithelium and endothelium is crucial for the development and function of most organs. Tight junction (TJ), the most apical element of epithelial and endothelial junctional complexes in vertebrates, is directly involved in this compartmentation by sealing cells to create the primary barrier to the diffusion of solutes through the paracellular pathway. TJ is also thought to function as a boundary between the apical and basolateral plasma membrane domains, which differ in protein and lipid compositions and physiological functions, to create and maintain epithelial and endothelial cell polarity. <br /> Other intercellular junctions such as adherens junctions, desmosomes, and gap junctions bear specific types of integral membrane proteins which play crucial roles in each junction. To clarify the structure and function of TJ at the molecular level, an integral membrane protein should be identified. In TJ, despite of intensive studies, an integral membrane protein remained elusive for quite some time. Here, by the use of the monoclonal antibody technique, he first identified an integral membrane protein localizing at TJ, which was designated as "occludin" . Then he cloned Its cDNA, which enabled us to clarify some unique functions of this molecule; its association with the some peripheral membrane proteins of TJ, its localization signal at TJ, and its cell adhesion ability. <br /> In the Chapter 1, he identified and integral membrane protein of TJ and analyzed its primary structure by cDNA cloning. Recently, we found that Z0-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M. , A.Nagafuchi, S.Yonemura, T.Kitani-Yasuda, Sa.Tsukita, and Sh.Tsukita. 1993. J.Cell Biol 121:491-502). Using this fraction derived from chick liver as an antigen, he obtained three monoclonal antibodies specific for a ～65kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this ～65kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: At the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, he cloned and sequenced its cDNA. He found that the cDNA encoded a 504 amino acid polypeptide with the calculated molecular mass of 55.9kD. A search of the data base identified no proteins with significant homology to this membrane protein. <br /> A most striking feature of its primary structure was revealed by a hydrophilicity plot: Four putative membrane-spanning segments were included in the N-terminal half. This hydrophilicity plot was very similar to that of connexin, an Integral membrane protein In gap Junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which they designated as 'occludin' . <br /> In the Chapter 2, he investigated the roles of the COOH-terminal cyto-plasmic domain of occludin using its cDNA. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the long COOH-terminal cytoplasmic domain consisting of 255 amino acids (domain E), especially its COOH-terminal ～150 amino acids (domain E358/ 504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in E.coli as a fusion protein with glutathione-S-transferase (GST), and this fusion protein was shown to be specifically bound to a complex of Z0-1 (220kD) and Z0-2 (160kD) among various membrane peripheral proteins. In vitro binding analyses using GST-fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the Z0-1/Z0-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant Z0-1 produced in E.coli. He concluded that occludin itself can localize at TJ and directly associate with Z0-1. The coincidence of the sequence necessary for the Z0-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through Z0-1 is required for occludin to be localized at TJ. <br /> In the Chapter 3, he investigated the cell adhesion ability of occludin. Chick occludin was overexpressed in insect cells by recombinant baculo virus infection. When the cells were observed by confocal immunofluores-cence microscopy, the majority of expressed chick occludin occurred inside cells. Thin section electron microscopy of these cells identified peculiar electron-dense membrane structures which consist of thin parallel or concentric lamellae. These multilamellar structures were shown to consist of many disk-like structures each of which has a loop of membrane with its both ends. These structures were heavily labeled with anti-chick occludin monoclonal antibodies. These observation led us to speculate that the disk-like structure was transformed from each cisterna whose luminal space was completely collapsed by accumulation of occludin molecules. Furthermore he analyzed the arrangements of intramembranous particles in the multilamellar structures by freeze-fracture technic. Two distinct types of fracture images were observed: In one type, numerous particles ～101μm in diameter were densely packed in a ramdom or linear pattern, whereas in the other type short straight grooves were occasionally observed. He concluded that under the condition of this study, occludin shows a tendency to polymerize into a short strand inside the membrane. These findings provided the first evidence that occludin is an adhesion molecule working at TJ in a homophylic manner. <br /> This study on identification and characterization of occludin, a novel adhesion molecule of TJ, opened a new way to analyze the structure and function of TJ at the molecular level. Further analyses of occludin will lead us to a better understanding how the permeability and polarization of epithelial and endothelial cell sheets are regulated in the near future. |
要旨・審査要旨 / Abstract, Screening Result (292.1 kB)
