Characterization of the post-translational modifications of the secreted acid phosphatase of Leishmania donovani
| dc.contributor.author | Lippert, Dustin Norman Dean | |
| dc.contributor.supervisor | Olafson, Robert Walter | |
| dc.date.accessioned | 2018-10-30T18:40:15Z | |
| dc.date.available | 2018-10-30T18:40:15Z | |
| dc.date.copyright | 2001 | en_US |
| dc.date.issued | 2018-10-30 | |
| dc.degree.department | Department of Biochemistry and Microbiology | en_US |
| dc.degree.level | Doctor of Philosophy Ph.D. | en_US |
| dc.description.abstract | The secreted acid phosphatase (SAcP) of Leishmania donovani is a secreted glycoprotein modified with unique glycoforms which share a structural heterogeneity and immunological similarity with the dominant, cell surface phosphoglycolipid produced by all species of leishmania parasites. The post-translational modifications of this enzyme are structurally diverse and include standard high-mannose type N-linked glycosylations as well as a novel O-linked phosphoglycan. This dissertation encompasses the analysis of these structures including assessment of their size, hexose composition and sites of attachment to the protein. These analyses have employed both carbohydrate and protein chemistry techniques, as well as physical methods such as mass spectrometry. The N-linked glycosylations have been compared with those previously characterized on other Leishmania proteins and show substantial structural similarity. The O-linked phosphoglycans are unique to L. donovani, and are composed of phosphodisaccharides with the structure 4-O-(beta-D-galactopyranosyl)-alpha-D-mannopyranosyl-1-phosphate. These phosphodisaccharides are arranged in linear polymers by way of a phosphodiester linkage between the C1 hydroxyl of mannose and the C6 hydroxyl of galactose. Linkage of this structure to the protein is novel and proceeds via a phosphodiester to selected serine residues that are contained within a consensus protein sequence. This sequence occurs in excess of 20 times within the SAcP, which results in abundant glycan modification and contributes to the heterogeneity displayed by this enzyme. The biosynthetic machinery used to produce these structures was also investigated. The addition of phosphoglycan to the SAcP is initiated by the transfer of alpha-D-mannopyranosyl-1-phosphate from GDP-Man to the protein catalysed by a mannosyl phosphate transferase (MPT). An assay for this enzyme is described using a synthetic peptide substrate to which radiolabeled mannose can be transferred from GDP-[14C] Man. This assay has been used to partially characteriz the MPT and has assisted in the isolation of the enzyme using an affinity chromatography approach. Sequence analysis and amino acid analysis of the enzyme isolated in this way has shown that the MPT is a novel molecule that does not presently exist in the public database. | en_US |
| dc.description.scholarlevel | Graduate | en_US |
| dc.identifier.uri | http://hdl.handle.net/1828/10201 | |
| dc.language | English | eng |
| dc.language.iso | en | en_US |
| dc.rights | Available to the World Wide Web | en_US |
| dc.subject | Leishmania | en_US |
| dc.subject | Enzymes | en_US |
| dc.subject | Glycoproteins | en_US |
| dc.title | Characterization of the post-translational modifications of the secreted acid phosphatase of Leishmania donovani | en_US |
| dc.type | Thesis | en_US |