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dc.contributor.authorBarnes, Jarrod Wesley
dc.date.accessioned2014-03-04T20:23:28Z
dc.date.available2014-03-04T20:23:28Z
dc.date.issued2011-12
dc.identifier.otherbarnes_jarrod_w_201112_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/barnes_jarrod_w_201112_phd
dc.identifier.urihttp://hdl.handle.net/10724/27664
dc.description.abstractThe mannose-6-phosphate (Man-6-P) targeting pathway represents one of the best-studied examples of the ability of carbohydrates to carry information. Acid hydrolases that are modified with Man-6-P residues on their N-glycans are targeted to the lysosomes by Man-6-P receptors. In addition to these enzymes, many non-lysosomal proteins have been shown to bear phosphomannosyl residues. The functional relevance of the mannose phosphorylation on non-lysosomal proteins, however, remains poorly defined. More importantly, the pathogenic consequences associated with the impaired mannose phosphorylation of these proteins in the lysosomal storage disease mucolipidosis II (ML-II) have not been determined. In the present work, the extent and functional role of mannose phosphorylation on two non-lysosomal proteins, leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGF-β1), was investigated using biochemical approaches. Secreted LIF was demonstrated to be highly Man-6-P modified by cation-independent Man-6-P receptor (CI-MPR) affinity chromatography. Removal of specific N-glycan sites on LIF resulted in decreased mannose phosphorylation and a corresponding increase in its steady-state extracellular levels. Subsequent experiments showed that LIF mannose phosphorylation controls its extracellular levels by means of Man-6-P-dependent internalization and direct intracellular targeting of this molecule to the lysosomes. Loss of mannose phosphorylation in ML-II likely leads to excessive extracellular LIF, providing a potential mechanism for the bone phenotypes associated with this disease. Contrary to our findings with LIF, latent TGF-β1 was demonstrated to be poorly mannose phosphorylated and its activation was not altered by free Man-6-P addition when latent forms were present. These results indicate that latent TGF-β1 activation is not mediated by direct Man-6-P modification of its latency-associated peptide. Although loss of mannose phosphorylation in ML-II was not predicted to alter TGF-β1 activation, decreased signaling of this growth factor, and a corresponding reduction in wound closure, was surprisingly noted in human and feline ML-II fibroblasts. Diminished activation of latent TGF-β1 was further associated with a striking decrease in its detergent solubility, possibly due to upregulation of tissue transglutaminase. Collectively, these results indicate that the loss of Man-6-P biosynthesis on non-lysosomal proteins may directly (LIF) or indirectly (TGF-β1) affect their function and contribute to the pathogenesis of ML-II.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectN-linked glycosylation
dc.subjectmannose-6-phosphate
dc.subjectnon-lysosomal protein
dc.subjectleukemia inhibitory factor
dc.subjecttransforming growth factor beta
dc.subjecttransglutaminase 2
dc.subjectmucolipidosis II
dc.titleFunctional role for the mannose phosphorylation on two non-lysosomal proteins
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorRichard Steet
dc.description.committeeRichard Steet
dc.description.committeeMichael Tiemeyer
dc.description.committeeWalter Schmidt
dc.description.committeeKelley Moremen


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