Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies.
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.
Abo
S
S
Neuroimmunology Laboratory, La Trobe University, Bundoora, Victoria, Australia.
Bernard
C C
CC
Webb
M
M
Johns
T G
TG
Alafaci
A
A
Ward
L D
LD
Simpson
R J
RJ
Kerlero de Rosbo
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002490:Central Nervous System / Q000737:chemistry; D003711:Demyelinating Diseases / Q000139:chemically induced; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009103:Multiple Sclerosis / Q000276:immunology; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D063308:Myelin-Oligodendrocyte Glycoprotein; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D009836:Oligodendroglia / Q000737:chemistry*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; C571326:MOG protein, human; D008562:Membrane Glycoproteins; C571328:Mog protein, rat; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D063308:Myelin-Oligodendrocyte Glycoprotein; D009419:Nerve Tissue Proteins