Expression of biologically active human corticosteroid-binding globulin and analysis of the steroid binding site by site-directed mutagenesis and chemical modification.
Item
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Title
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Expression of biologically active human corticosteroid-binding globulin and analysis of the steroid binding site by site-directed mutagenesis and chemical modification.
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Identifier
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AAI9304667
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identifier
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9304667
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Creator
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Ghose-Dastidar, Jayasri.
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Contributor
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Advisers: J. B. Alexander Ross | Reza Green
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Date
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1992
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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 | Biophysics, Medical
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Abstract
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Human corticosteroid binding globulin (hCBG) is a serum glycoprotein that binds the steroid hormones cortisol and progesterone with high affinity. It appears that hCBG may have important physiological functions, such as delivery of steroid to sites of inflammation. To understand the biogenesis of hCBG and to analyse its steroid binding site, the cDNA sequence has been isolated, and expressed in the baculovirus/insect cell system. Quantitation of the expressed hCBG secreted by these cells shows that high levels of biologically active recombinant hCBG can be produced in this system ({dollar}\approx{dollar}50 pmol/2.5 {dollar}\times{dollar} 10{dollar}\sp6{dollar} cells derived medium). Thus this system should be useful for producing sufficient hCBG for physico-chemical analysis. Further characterization of this recombinant hCBG shows that, although the secreted hCBG is high mannose containing, it has identical affinities and specificities for different steroids as that of native hCBG. This indicates that processing of core-glycosylated hCBG to complex type carbohydrate containing species in the Golgi compartment is not necessary for hCBG to achieve a steroid binding conformation. Instead, the binding properties of unglycosylated hCBG produced in insect cells, demonstrate that the process of N-linked glycosylation is crucial for hCBG to attain its biologically active form. However, N-glycosylation is not the sole determinant of the active conformation, since the in vitro translated, N-glycosylated hCBG does not bind cortisol. It thus appears that intracellular N-glycosylated hCBG undergoes some further structural maturation during intracellular transport.;To address the possible role of specific cysteine residues in the steroid binding process (as suggested by previous studies), mutant hCBGs containing serine or alanine in place of cys{dollar}\sb{lcub}228{rcub}{dollar}, have been produced by site-directed mutagenesis and construction of corresponding recombinant baculoviruses. Scatchard analysis shows that alteration of cys{dollar}\sb{lcub}228{rcub}{dollar} to serine or alanine does not change the steroid binding affinity of hCBG, demonstrating that cys{dollar}\sb{lcub}228{rcub}{dollar} is not involved in the binding interaction. This finding leads to the hypothesis that cys{dollar}\sb{lcub}60{rcub}{dollar} may be the functionally important cysteine. By modifying the wild-type and mutant hCBGs with the sulfhydryl-specific reagents N-ethylmaleimide (NEM), iodoacetamide (IAA), and sodium tetrathionate, it has been demonstrated that cys{dollar}\sb{lcub}60{rcub}{dollar} is present at the steroid binding site and may possibly be involved in the steroid binding process. Requirement of dithiothreitol treatment for achieving complete inactivation of binding activity by NEM and sodium tetrathionate strongly indicates that cys60 is the accessible cysteine which is partially oxidized under native conditions.
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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.
