Structural and functional characterization of the secreted acid phosphatase produced by Leishmania donovani

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dc.contributor.authorSigurdson, Andrea Gailen_US
dc.date.accessioned2024-08-15T18:23:00Z
dc.date.available2024-08-15T18:23:00Z
dc.date.copyright1992en_US
dc.date.issued1992
dc.degree.departmentDepartment of Biochemistry and Microbiologyen_US
dc.degree.levelMaster of Science M.Sc.en
dc.description.abstractSecreted acid phosphatase (sACPase) was purified from the supernatants of in vitro Leishmania donovani 1S2D cultures. The sACPase was found to be a high molecular weight polydisperse glycoprotein species having a strong net negative charge. The N-terminal sequence of the sACPase protein was determined and compared to previously reported sequences of Leishmania sACPases. L. donovani lipophosphoglycan (LPG)-specific monoclonal antibodies (mAbs) were tested for binding to the sACPase. MAbs L98 and L157, specific for the LPG-associated protein did not bind the sACPase, nor did the sACPase-specific mAb 16Al cross-react with LPG; however, the anti-LPG phosphodisaccharide mAb CA7AE bound the sACPase, indicating the presence of an LPG-like [-PO₄-6Galactose (βl-4)Manoseαl-] epitope on the sACPase. Enzyme activity trapping assays and immunoblots using mAb CA7AE demonstrated that the phosphodisaccharide epitope of the sACPase was very strongly associated with the enzyme. Experiments were performed to characterize the nature of the linkage between this unusual phosphodisaccharide structure and the sACPase protein. Growth of L. donovani promastigotes in the presence of asparagine-linked glycosylation inhibitors did not eliminate the phosphodisaccharide epitope. Digestion of native sACPase with endo-β-N-acetylglucosaminidases also failed to remove the phosphorylated structure. These data suggested that the phosphodisaccharide was not attached to the protein through an N-linked oligosaccharide structure, although the actual mode of attachment was not determined. SACPase produced by promastigotes cultured in the presence of N-glycosylation inhibitors showed no enzyme activity, indicating that glycosylation may be required for enzyme activity. Endoglycosidase-mediated removal of the N-linked oligosaccharides and exoglycosidase-mediated cleavage of mannose residues from native sACPase caused a gradual loss of enzymatic activity. This suggested that the N-glycosylations were essential for the maintenance of the enzyme activity even after synthesis and secretion. This role of the oligosaccharides was not altered when Leishmania promastigotes were grown in the presence of glycosylation processing inhibitors, nor was it affected by the presence or absence of the phosphodisaccharide structure. The far ultraviolet circular dichroism spectra of native and endoglycosidase-digested sACPase indicated possible changes in the secondary or tertiary structure of the protein upon deglycosylation, providing preliminary evidence for a role of the sACPase N-linked oligosaccharides in the maintenance of the active protein conformation. Potential physiological roles for the sACPase were studied through characterization of the substrate specificity of the enzyme. The sACPase was found to dephosphorylate various inositol phosphates at a rate that may be physiologically significant. In addition, mannose-6-phosphate was identified as an optimal monophosphorylated monosaccharide substrate for the sACPase. The enzyme showed some activity toward phosphorylated amino acids, but did not hydrolyze a phosphopeptide substrate. Together, these data suggest that the L. donovani sACPase may be an important factor in the host-parasite relationship that is established during Leishmania infection.en
dc.format.extent106 pages
dc.identifier.urihttps://hdl.handle.net/1828/19689
dc.rightsAvailable to the World Wide Weben_US
dc.subjectUN SDG 15: Life on Landen
dc.titleStructural and functional characterization of the secreted acid phosphatase produced by Leishmania donovanien_US
dc.typeThesisen_US

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