Mapping the functional regions of the myelin Po protein's extracellular domain.
Item
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Title
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Mapping the functional regions of the myelin Po protein's extracellular domain.
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Identifier
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AAI9830784
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identifier
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9830784
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Creator
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Zhang, Kejia.
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Contributor
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Adviser: Marie T. Filbin
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Date
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1998
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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, Neuroscience | Biology, Molecular | Biology, Cell
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Abstract
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Po, the most abundant protein of peripheral nervous system (PNS) myelin is a homophilic adhesion molecule and is directly involved in the compaction of PNS myelin. To map more precisely regions of Po's extracellular domain that are critical for adhesion, antibodies against three hydrophilic sequences, Po 38-46, 74-82 and 91-95 and the peptides themselves were assessed for their ability to inhibit the adhesion of Po-expressing CHO cells. Both peptide 91-95 and its antibody completely inhibited the aggregation of Po-expressing cells. In addition, peptide 74-82 and its antibody also inhibited adhesion but not as effectively as peptide 91-95. In contrast, neither peptide 38-46 nor its antibody had any effect on Po-mediated adhesion. This suggests that sequence 91-95 in Po's Ig domain may be directly involved in the adhesion of Po, while the amino acid sequence 74-82 may be partially involved. To study further Po peptide 91-95, two amino acids, Asp92 and Gly94, conserved in a subset of Ig-like molecules, were replaced, each with a conservative substitution. Unlike the cells expressing the wildtype Po, cells expressing Po mutated at Asp92 and Gly94 did not form large aggregates. This shows that Po mutated at Asp92 and Gly94 is not capable of homophilic adhesion.;To address the role of the disulfide bond in an Ig-like domain, the disulfide bond between Cys21 and Cys98 of Po's Ig-like domain was prevented from forming by replacing Cys21 with alanine. The Cys21-mutated Po cDNA was expressed in CHO cells which, unlike the wildtype Po, failed to adhere. This suggests that the Po protein, when mutated at Cys21, does not behave like a homophilic adhesion molecule, which in turn implies that the formation of an Ig-like domain disulfide bond is essential to the functioning of Po.;To test the possible dominant negative effect of the aberrant Cys21 mutated Po on the adhesion of the wildtype Po, the cDNA of the Cys21-mutated Po was transfected into a CHO cell line already expressing wildtype Po. Two clones, each expressing both Cys21 Po and wildtype Po lost their Po-dependent adhesive properties, while a third clone, expressing only the wildtype Po, retained its adhesive properties. These results suggest that the presence of Cys21-mutated Po abolishes the adhesion of the wildtype Po protein.
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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.
