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Cloning and characterisation of a novel mitochondrial autoantigen associated with multiple sclerosis

Sanati, Mohammad Hossein (1996) Cloning and characterisation of a novel mitochondrial autoantigen associated with multiple sclerosis. PhD thesis, Murdoch University.

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Multiple Sclerosis (MS) is a chronic neurological disease of the central nervous system, characterised by a cellular immune response in the early stages followed by demyelination. Although the cause of MS is unknown, there is much evidence that points to MS as an autoimmune disease. To test the hypothesis that an autoantigen is involved in MS, we screened a Xgtl 1 human foetal spinal cord cDNA library using IgG 6 from patients with MS. From screening 2x10 recombinant phage, 6 positive clones were identified. Proteins expressed by these phage on bacterial lysate plates appeared to react specifically with pooled MS IgG but not with pooled IgG from normal human sera. The positive clones were amplified by the polymerase chain reaction and sequenced. After searching GenBank, analysis of the sequences showed a high percent similarity between three clones and the mitochondrial gene encoding the ND4 component of human NADH:ubiquinone reductase (Complex I).

Because the region cloned was associated with an active site in Complex I, submitochondrial particles were purified from different sources and the inhibition of Complex I was tested in presence of MS IgG. The pooled MS IgG strongly inhibited Complex I activity in sub-mitochondrial particles with decylubiquinone as a substrate. A peptide CLANSNYERTHSR, which is part of the ND4 protein in Complex I, was conjugated with a maleimido-thiol bond to diphtheria toxoid and used as an autoantigen and as an immunogen in a rabbit. To remove any IgG which bound to diphtheria toxoid and the bovine serum albumin blocking agent in the ELISA, sera were pre-adsorbed before being incubated with the peptide conjugate. About 20 % of patients with MS had antibody to the peptide and when present, the level was found to fluctuate. This is the first time that a mitochondrially encoded protein has been shown to be an autoantigen. Autoantibodies to the ND4 peptide were found in patients with Leber’s Hereditary Optic Neuropathy (LHON), which is of great interest as the commonest mutation site in this mitochondrial disease is adjacent to the epitope. In preliminary experiments the autoantibodies were also found in association with other autoantibodies in diseases where there is known to be extensive damage to cells.

There is a clear molecular mimicry between the ND4 epitope and a predicted epitope in a plasmid which encodes a surface protein in spirochaetes. Cross reaction between the spirochaete protein and the ND4 peptide was demonstrated by using the rabbit antibody to ND4.

While the most likely explanation for the presence of the autoantibody to Complex I is that it is a marker of damage to mitochondria in MS, it may have a secondary role in the pathogenesis if a spirochaete triggers an autoimmune response to cells presenting peptides from damaged mitochondrial proteins on their surface.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Carnegie, Patrick
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