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Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for
1Neuroimmunology Laboratory, La Trobe University, Bundoora, Victoria, Australia.
Abstract:
Myelin oligodendrocyte glycoprotein, (MOG), a quantitatively minor central nervous system (CNS) myelin component, is a candidate target antigen for autoimmune-mediated demyelination. It is a highly hydrophobic protein present in very small amounts in CNS tissue and thereby difficult to purify. Our aim was to devise a purification procedure that would yield sufficient quantities of highly purified MOG to subsequently test its potential encephalitogenic activity, as well as investigate the humoral and cell-mediated responses to this antigen in naturally occurring and experimentally induced autoimmune demyelinating diseases. MOG was purified from human CNS white matter using immunoaffinity chromatography, a procedure that gave a final yield of MOG corresponding to 0.02% total white matter protein. The final product, which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as shown also by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for myelin basic protein, proteolipid protein and myelin-associated glycoprotein. Partial amino acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto PVDF. The sequence of the first 17 N-terminal amino acids had approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences obtained showed also very high homology. Our purified MOG preparations have been used to investigate T cell response to MOG by peripheral blood lymphocytes of multiple sclerosis patients and to induce a relapsing remitting demyelinating disease in Lewis rats.
Insights
Researchers purified myelin oligodendrocyte glycoprotein (MOG) from human brain tissue. This highly pure MOG was used to study autoimmune demyelinating diseases like multiple sclerosis.
Area of Science:
- Neuroscience
- Immunology
- Biochemistry
Background:
- Myelin oligodendrocyte glycoprotein (MOG) is a key component of central nervous system (CNS) myelin.
- MOG is implicated as a potential target antigen in autoimmune demyelinating diseases.
- Its hydrophobic nature and low abundance in CNS tissue present purification challenges.
Purpose of the Study:
- To develop a purification method for obtaining sufficient quantities of highly purified MOG.
- To investigate the encephalitogenic potential of MOG.
- To analyze humoral and cell-mediated immune responses to MOG in autoimmune demyelinating conditions.
Main Methods:
- Immunoaffinity chromatography was employed to purify MOG from human CNS white matter.
- Purified MOG was analyzed for purity using SDS-PAGE and immunoblotting with specific antibodies.
- Partial amino acid sequencing was performed on purified MOG bands.
Main Results:
- A purification procedure yielded MOG at 0.02% of total white matter protein.
- The purified MOG showed high purity, migrating at 28 kDa and 58 kDa, with specific reactivity to anti-MOG antibodies.
- Partial amino acid sequences exhibited high homology to rat MOG sequences.
- Purified MOG induced relapsing-remitting demyelinating disease in Lewis rats and was used to study T cell responses in multiple sclerosis patients.
Conclusions:
- A robust method for MOG purification was established, yielding highly pure protein.
- The purified MOG serves as a valuable tool for studying autoimmune demyelination.
- MOG is confirmed as an encephalitogenic antigen, relevant to multiple sclerosis pathogenesis.