The bovine spliceosomal U1 small nuclear ribonucleoprotein particle : a study of its autoantigenicity and biochemical properties : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
Despite individual autoimmune diseases being relatively rare, collectively these diseases afflict 8 % of the population according to the American Autoimmune Related Diseases Association. With over 75 % of those affected being women, autoimmune disease has been recognised, by the World Health Organisation and the US National Institutes of Health, as a major global women's health issue. One third of autoimmune sufferers have a rheumatological disorder, which commonly affect the joints, muscle, skin, salivary glands and kidneys. Antibodies against nuclear antigens are a serological hallmark of these diseases. Detection of these antibodies is used in the diagnosis and prognosis of the disease. The sensitivity and specificity of the test, of which the antigen is a key component, is pivotal to correct disease diagnosis and management. The relationship between circulating autoantibodies and the target antigen is complex. Improving the effectiveness of a test to assist in diagnosis and prognosis comes from characterisation and understanding these complex relationships. This thesis compares bovine spliceosomal U1 small nuclear ribonucleoprotein particle (U1 snRNP) complex with its human equivalent, and examines the validity of using this bovine derived autoantigen in the diagnosis of the human autoimmune diseases, systemic lupus erythematosus and mixed connective tissue disease. Differences between bovine and human U1 snRNP composition were characterised using a combination of electrophoretic, immunoassay and mass spectrometry techniques. Although the U1C protein could not be identified in bovine U1 snRNP, all other specificities were present. U1A remained intact, whilst the U1 snRNP specific 68K protein was dephosphorylated and a large C-terminal domain was removed, such that 68K migrated as a 30-36 kDa cluster on SDS-PAGE. Bovine SmD proteins, present in U1 and non-U1 snRNPs, were unaffected, whereas, SmB'/B was truncated to a 12 kDa peptide, which interestingly, was no longer reactive with anti-RNP sera in western blot. The recognition of human SmB'/B protein by anti-RNP sera in western blot was further examined. A technique was developed to immunoaffinity purify tryptic digests of SmB'/B which could then be analysed by mass spectrometry. Interestingly, the human replication element protein (HREP) was tentatively identified, rather than SmB'/B as expected. It may be possible, therefore, that anti-RNP sera may be reacting with a protein other than SmB'/B. To examine the contribution of the individual U1 snRNP proteins to anti-RNP and anti-Sm sera reactivities, a method was developed to dissociate bovine U1 snRNP and to purify the individual component antigens. It was demonstrated both empirically and through anecdotal feedback from a commercial diagnostic kit producer that patient sera respond better to purified Sm-free 68K than the recombinant 68K antigen. The effect of commercial processing of bovine thymus, the source for U1 snRNP antigen, was determined. In this study, variables that may be controlled during processing, such as temperature, protease activity and pH, were investigated. Hydrolysis of the intact human 68K protein with the necrotic protease, cathepsin L, produced 38 and 25 kDa fragments, whereas exposure to ambient temperature and low pH produced 32 kDa peptide fragments similar to those observed in purified bovine 68K. It was therefore proposed that 68K protein may undergo autocatalytic hydrolysis during necrotic cell death. Thorough characterisation of the bovine spliceosomal U1 snRNP proteins has not only validated their use as diagnostic reagents in autoimmune disease but also provided some insight into the inactivation of U1 snRNP function during early cell death.