Molecular analysis of MPO for patient with autoimmune disease ANCA

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In pauci-immune necrotizing crescentic glomerulonephritis (NCGN) autoimmune disease, anti myeloperoxidase (MPO) -antibody increase in peripheral blood of the patients.Determination of epitopes of anti-MPO antibody in some autoimmune diseases such as NCGN my give us a clue to the etiology and be used for prediction of the diseases.We have demonstrated three types of MPO (I,II,and III) and that some sera of GN reacted with the large subunit (59 kDa) of MPO-III,but not to the small subunit (14 kDa).As one serum of them most strongly reacts with a 55-kDa-deglycosylated form derived from the 59-kDa large subunit, we have attempted to prepare some recombinant the large fragments of large subunit.In the present study, we prepared the recombinant MPO-fragments as panels to analyze epitope of serum of patient with autoimune disease.cDNA encoding 59 kDa-subunit (large) of MPO was amplified by PCR in 12 parts and these cDNAs were separately inserted into the vector.The recombinant MPO fragments were expressed in E.coli and were digested with guanidine hydrochloride in a sonicator.The fragments were purified with an affinity column chromatography.The obtained fragments were used for western blot analysis to determine the reactivity of sera of patients.Some sera reacted with the MPO fragments containing Met409.The results confirmed the recognition site in native MPO-III molecule by an anti-MPO serum is around sugar attachment sites in the N-terminus in the large subunit of MPO.