| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Cell polarization is an important character on tissue formation of biological functions. To clarify whether N-linked sugar chains are related to cell polarization, Madin-Darby canine kidney (MDCK) cells polarized on membrane filter were used as the model. First, the lectin a affinity against proteins secreted from apical and basolateral sides of [^<35>S]methionine labeled cells were analyzed. These results showed that the binding against Con A and AAL columns was great different between apical and basolateral proteins, indicationg that secreted glycoproteins from apical side have distinct sugar chain compositions from those of basolateral side. Second, the sugar chain structures of membrane-bound glycoproteins from each side were analyzed. After labeling polarized MDCK cells with [^3H]mannose, membrane-bound glycoproteins of each side were biotinylated, lysed with trition X-100 and purified through avidin column.
After [^3H] mannose-labeled oligosaccharides were released from these glyc
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oproteins by hydrazinolysis, they were analyzed with paper electrophoresis, sialidase digestion and methanolysis. These results showed that the ratio of neutral sugar chains : sialylated sugar chains : sulfated sugar chains of both sides were about 20 : 3 : 1. On Bio-Gel P-4 column chromatography and severallectin column chromatographies, the neutral sugar chains were mainly high mannose type and acidic sugar chains were complex type including N-acetyllactosamine repeating structures. It was also elucidated that apical membrane glycoproteins were more fucosylated than basolateral ones. On the other hand, transport vesicle protein VIP 36 was analyzed because the N-terminal encoded domain shows homology to leguminous plant lectins. The cDNA encoded extracelldomain of VIP 36 was prepared from total RNA of MDCK cells by the method of RT-PCR,and linked to the downtream of cDNA encoded gluthatione-S-transferase (GST) in pGEX.The plasmids were transformed in E.coli, and the fusion proteins (GST / Vip36) were expressed and purified with gluthatione Sepharose 4B.The purified proteins were used for examination of binding to [^<35>S]methionine labeled apical secreted proteins. It was found that the fusion proteins bind several secreted proteins under the condition of pH 5.5 and they dissociate under pH 7.4. The relationship of VIP 36 and fucosylated sugar chains found in mainly apical membrane proteins is now investigated. Less
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