Ceruloplasmin oxidizes the toxic ferrous (Fe(II)) form of iron to the ferric (Fe(III)) form, and promotes iron loading onto transferrin. It is produced by the liver and secreted into the plasma. My work has shown that a novel membrane-anchored form of ceruloplasmin is the major form of this enzyme in the rat brain. / The membrane-anchored form of ceruloplasmin was identified using a monoclonal antibody (mAb 1A1) that specifically labels the surface of astrocytes. Using mAb 1A1 immunoaffinity chromatography, the antigen was purified from detergent extracts of rat C6 glioma cells. Amino acid microsequencing, and additional experiments, revealed that this antigen was highly similar to ceruloplasmin. Further experiments revealed that this form of ceruloplasmin was directly anchored to the surface of astrocytes and C6 glioma cells by a glycosylphosphatidylinositol (GPI) anchor. / Screening of a C6 glioma cDNA library identified a novel alternatively-spliced transcript that codes for this GPI-anchored form of ceruloplasmin. HEK293T cells transfected with the novel full-length cDNA expressed ceruloplasmin on the cell surface. Treating these transfected cells with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves GPI anchors, eliminated the cell surface localization of ceruloplasmin, confirming that the novel cDNA codes for the GPI-anchored form of the protein. RNase protections on rat brain and liver RNA revealed that GPI-anchored ceruloplasmin is the major form in the brain, whereas the liver expresses predominantly the secreted form. / To better understand the role of ceruloplasmin in vivo, a ceruloplasmin gene knockout mouse was generated. The knockout mice accumulate large amounts of iron in the liver and have reduced serum iron levels. Older knockout mice (15--17 months) have increased iron accumulation in different parts of the CNS, including the cerebellum, spinal cord, and retina. Increased lipid peroxidation is also observed in some regions of the CNS. These biochemical changes were accompanied by deficits in motor skills. Furthermore, dissociated cell cultures of the cerebellum from knockout mice were more susceptible to free radical injury when treated with hydrogen peroxide. These data suggest that ceruloplasmin plays an important role in iron metabolism in the CNS and provides protection against free radical injury.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37807 |
| Date | January 2000 |
| Creators | Patel, Bharatkumar N. |
| Contributors | David, Samuel (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Neuroscience.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001803226, proquestno: NQ70123, Theses scanned by UMI/ProQuest. |
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