Human acid beta-glucosidase: Investigation of N-glycosylation through mutagenesis and heterologous expression.
Item
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Title
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Human acid beta-glucosidase: Investigation of N-glycosylation through mutagenesis and heterologous expression.
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Identifier
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AAI9405499
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identifier
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9405499
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Creator
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Berg-Fussman, Anat.
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Contributor
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Advisers: Gregory A. Grabowski | Robert J. Desnick
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Date
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1993
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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, Genetics | Biology, Cell | Chemistry, Biochemistry
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Abstract
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Deficient activity of human acid {dollar}\beta{dollar}-glucosidase, a lysosomal membrane glycoprotein, causes the most prevalent lysosomal storage disease, Gaucher disease. To better understand the etiology of this autosomal recessive disorder and to be able to design effective therapies, it is important to determine the structure/function relationship of human acid {dollar}\beta{dollar}-glucosidase. To define N-glycosylation site occupancy and to gain insights into its role(s) for human acid {dollar}\beta{dollar}-glucosidase's normal function, the five potential N-glycosylation sites (sequons) were mutated individually and collectively.;Utilizing site-directed in vitro mutagenesis, each and all of the sequons (Asn-Xaa-(Ser/Thr)) were eliminated by aspargine(N) to glutamine(Q) substitutions. Heterologous expression in insect (Sf9) and COS-1 cells, followed by comparative SDS-PAGE analysis, identified that the occupied sequons were located at N19, N59, N146 and N270. The sequon located at N462 was not occupied. The occupancy of the first site was critical for the development of an active acid {dollar}\beta{dollar}-glucosidase. Additional mutations, N19D, N19E and T21G were created within the first site. N19D exhibited normal catalytic activity and increased activation by phosphatidylserine. However, significant but variable, decreased enzymatic activities were observed with N19E and T21G. The N59Q, N146Q and N270Q substitutions had normal kinetic and catalytic properties.;The results of these studies demonstrate the importance of Asn-linked carbohydrates at the first site, and/or the nature of amino acid at residue 19, for the development of a catalytically active human acid {dollar}\beta{dollar}-glucosidase form. This is the first demonstration of a lysosomal hydrolase that requires a specific sequon occupancy for its catalytic activity. The results of the heterologous expression studies of these mutants may contribute to the future design of improved therapeutic proteins for Gaucher disease.
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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.
