THE THYROPEROXIDASE DOUBLET IS NOT PRODUCED BY ALTERNATIVE SPLICING

Thyroperoxidase is a membrane-bound, heme-containing enzyme which cata lyses iodination of thyroglobulin and coupling of resulting iodotyrosi nes to produce thyroid hormone. In addition to the full length molecul e of 933 amino acids (TPO1), Northern blotting and sequencing have rev ealed several shorter transcripts. The most abundant is a species lack ing 171 nucleotides in which the alternative splicing results in the d eletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcr ipts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 5 7 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Bot h recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification f rom a bacterial lysate on an amylose column. Rabbits have been immuniz ed by intradermal injection of 500 mu g of fusion protein, initially i n complete Freund's adjuvant followed by two boosts, at 2-week interva ls, in incomplete Freund's adjuvant. The resulting high titre immune s era (IS) were reactive with the relevant immunising antigens, when tes ted by ELISA. Depletion of each serum by passage through an MBP-CNBr S epharose column allowed purification of antibodies against the relevan t peptides, as demonstrated by ELISA with the appropriate fusion prote in and MBP. This demonstrates that we have produced specific polyclona l antibodies for the 57 amino acids unique to TPO1 and for the amino a cid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and G raves' thyroid membranes, in reducing and non-reducing conditions. Mon oclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reduci ng conditions, we observed a broad signal at 105-110 kDa, which appear ed to comprise two bands, with both polyclonal antibodies and the mono clonal. There was no difference in the image between the normal and th e Graves' thyroid. In reducing conditions, the broad signal resolved c learly into two distinct bands, one at 105 and the other at 110 kDa. O nce again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that t he TPO doublet is not the consequence of translation of TPO2.