Title

Myelin protein and lipid sorting mediated by glycosphingolipid-enriched microdomains

Date of Completion

January 1998

Keywords

Biology, Neuroscience|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Oligodendrocytes (OLs) produce remarkable quantities of proteins and lipids during myelin biogenesis. To target them precisely to specific domains of myelin sheaths, OLs must utilize a highly regulated sorting and transport system. A better understanding of myelin protein and lipid sorting mechanisms should furnish information for the development of treatments for demyelinating diseases.^ To study sorting in myelin biogenesis, this thesis is focused on the "lipid rafts" hypothesis, which states that certain proteins are co-sorted with glycosphingolipid-enriched microdomains in the trans-Golgi network. The enrichment of galactocerebroside and sulfatide in OLs/myelin led me to hypothesize that OLs utilize lipid rafts in myelin biogenesis. To test this hypothesis: (1) Detergent-insoluble glycosphingolipid-enriched complexes (DIGs) were isolated from myelin and analyzed to identify proteins associated with lipid rafts. A protein localized in the paranodal loops, $2\sp\prime,3\sp\prime$-cyclic nucleotide 3$\sp\prime$-phosphodiesterase was associated with DIGs, whereas the major compact myelin proteins myelin basic protein and proteolipid protein, and the periaxonal protein myelin-associated glycoprotein were not. (2) Myelin DIGs isolated from knock-out mice lacking UDP-galactose: ceramide galactosyltransferase (CGT), the enzyme responsible for the synthesis of galactocerebroside, were compared to those from wild type animals. Several unidentified myelin proteins present in normal myelin DIGs were absent in DIGs from CGT-deficient mice. (3) MVP17, a highly-enriched protein in DIGs from OLs/myelin was cloned and characterized. The expression of MVP17, a novel tetraspan myelin membrane protein, was developmentally regulated and parallels the time-course of active myelination. Recombinant MVP17 with an epitope tag showed a punctate staining pattern in the cell body, processes and membranes of transfected cells.^ The presence of myelin proteins associated with DIGs provides a clue for the involvement of lipid rafts in myelin biogenesis, presumably in a domain-specific manner. The different protein composition of glucocerebroside-rafts (i.e., CGT-deficient mice) compared with galactocerebroside-rafts suggests that proteins are differently sorted based on the character of associated lipids. In summary, this thesis provides a new direction for studying protein-lipid sorting in myelin biogenesis based on a novel hypothesis of the domain- and lipid-specific myelinogenic rafts formation. ^