Studies on the biosynthesis and secretion of decorin : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University],
[2000]
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| Subjects: |
| Abstract: | ABSTRACT: Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms : a proteoglycan substituted with a single chondroitin sulfate chain and two or three asparagine-linked oligosacharides ; and a core protein glycoform substituted with asparagine-linked glycans but devoid of chondroitin sulfate. Sequencing of the core protein glycoform shows that the serine in the glycosamineoglycan attachment site has not been modified, indicating that the first xylose of the chondroitin sulfate linkage region is not present. Addition of benzyl-β-D-xyloside stimulated sulfate incorporation indicating that the activity of the glycosyltransferases, subsequent to the xylosyltransferase, were not rate limiting. A UDP-xylose : core protein β-D-xylosyltransferase activity assay showed that the decorin core protein purified from eukaryotic cells is 10-fold more efficient substrate that a synthetic peptide of E. coli produced decorin. In the presence of excess core protein acceptor, xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis and the conformational information present in the core protein is critical to effective substrate recognition. Decorin can be efficiently secreted with asparagine-linked oligosaccharides alone or with a single chondroitin sulfate chain alone, however there is severely impaired secretion of core protein devoid of any glycanation. Decorin core protein mutant devoid of asparagine-linked oligosaccharide attachment sites will not be secreted by CHO cells deficient in xylosyltransferase, or by parental CHO K1 cells if the xyloseyltransferase recognition sequence is disrupted. This data suggests that quality control mechanisms sensitive to an absence of asparagine-linked oligosaccharides can be abrogated by interaction of the core protein with glycosaminoglycan synthetic machinery. The xylosyltransferase from Swarm rat chondrosarcoma could be a soluable enzyme and enzyme activity was maximal at pH 6.5 and 25 ̊C without divalent metal ions. The enzyme activity was quantitatively retained not only on Mono Q and hydorxyapatite column at pH 8.0 but also on heparin agarose and ConA column at pH 6.5. The xylosyltransferase activity was also bound to decorin core affinity column with 1 mM UDP. UDP-xylose binds specificaly to 35 kDa and 28 kDa proteins in a fraction containing enzyme activity. An understanding of this observation may lead to a better approach for purifying xylosyltransferase. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment for the requirements for the degree of Doctor of Philosophy December 2000." Approved as to style and content by: David J. McQuillan, Magnus Höök, Richard Sinden, Frederick Gimble, Fuller W. Bazer. |
| Physical Description: | xiii, 169 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 142-168). |