Functional characteristics of myelin Po protein mutations associated with Charcot -Marie -Tooth disease.
Item
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Title
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Functional characteristics of myelin Po protein mutations associated with Charcot -Marie -Tooth disease.
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Identifier
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AAI9946191
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identifier
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9946191
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Creator
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Li, Wenhui.
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Contributor
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Adviser: Marie T. Filbin
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular | Biology, Neuroscience | Health Sciences, Dentistry
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Abstract
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Po, the most abundant protein of peripheral nervous system (PNS) myelin, is responsible for compaction of PNS myelin. The functional significance of this molecule is also manifest in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth (CMT) 1B (and its variants), as mutations in the Po gene are reported to be associated with this disease. However, despite the rapid progress in identification of those mutations in recent years, little is known about the disease mechanism. Since most CMT1B patients are heterozygous for mutations in Po, the aberrant Po may not reach myelin. Then the disease would be caused by insufficient Po. Alternatively, the mutated Po protein may reach myelin but could have a dominant-negative effect on the wild type Po.;Using an in vitro transfection/adhesion assay, we have shown that Po behaves like a homophilic adhesion molecule. This assay also enables us to test how individual mutations affect the functioning of Po. Three mis-sense mutations (Ser34 mutated to Cys (S34Q, Asp6l mutated to Glu (D61E) and Lys67 mutated to Glu (K67E)), one deletion (Ser34 (DeltaS34)) and two non-sense mutations (Tyrl25 to stop codon (Y125X) and Tyr152 to stop codon (Y152X)), which each corresponds to a mutation found in patients, were created in Po cDNA and then transfected into CHO cells. We showed that, except for Y125X and Y152X for which no Po protein was detected, all other mutated Po proteins reached the cell surface but failed to adhere. This suggests that these mutations abolish the adhesive properties of Po. From the three dimensional model of the extracellular domain of Po based on the crystal structure, it is possible that mutations at Asp6l and Lys67 affect the binding site directly while deletion of S34 could disrupt the protein conformation. Mutation of Ser34 to Cys could involve both possibilities.;As for the two non-sense mutations (Y125X and Y152X), as no protein was expressed, insufficient Po in myelin must be the cause of the disease. For the other four mutations, their ability to cause a dominant-negative effect on wild type Po was tested by co-expression of wild type and mutated Po in the same cell. The adhesive properties of these co-expressors were compared to those of cells expressing only the wild type Po. It was found that these mutated Po proteins all exerted a dominant-negative effect on the wild type of Po, however the severity of the effect varied from mild to blocking adhesion of wild type Po completely. These results suggest that the adhesion of wild type Po protein is partially or completely abolished by the presence of those mutated Po proteins. Furthermore, there is a correlation between the mutation and the disease phenotype. That is to say, mutation of Ser34 to Cys is shown to have the strongest dominant-negative effect of all the mutations tested, which correlates with its more severe disease phenotype. In contrast mild CMTlB phenotypes are associated with all the other mutations studies. This indicates that this assay system can be used as an indicator of the disease phenotype predicted from different Po mutations.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
