plays a key role in the biosynthesis of thyroid hormones (Alex- ander ..... Johnson,K.R., Nauseef,N.M., Care,A., Wheelock,M.J., Shane,S., Hudson,S.,. Koeffler,P.
The EMBO Journal vol.6 no.13 pp.4193-4196, 1987
Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules
Frederick Libert', Jean Ruell, Marian Ludgate', Stephane Swillens1, Nicholas Alexander1'3, Gilbert Vassart"2 and Christiane Dinsart1 1Institut de Recherche Interdisciplinaire and 2Service de Gdndtique Medicale, Faculte de Medecine, Universitd Libre de Bruxelles, Campus Erasme, 808 route de Lennik, 1070 Bruxelles, Belgium 3Permanent address: University of California, San Diego, CA, USA Communicated by A.Burny
A Xgtll cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene. Key words: autoimmunity/microsomal antigen/sequence homology/thyroid peroxidase Introduction Thyroperoxidase (TPO) is a membrane-bound hemoprotein which plays a key role in the biosynthesis of thyroid hormones (Alexander, 1977; Taurog, 1986; Virion et al., 1985). It catalyzes both the iodination of the precursor protein thyroglobulin and the intramolecular coupling of specific iodotyrosines into thyroxine and triiodothyronine. For many years TPO has been difficult to purify because of its particulate nature. Preparations with high specific activity have been isolated only after releasing first the enzyme from the membrane by proteolysis, usually combined with detergent treatment (Alexander, 1977; Taurog, 1986; Virion et al., 1985). More recently, human TPO was purified in its native state by monoclonal antibody-assisted chromatography and appeared as two contiguous bands in the 100-kd region on SDS -PAGE (Czarnocka et al., 1985; Ohtaki et al., 1986). Other experiments with monoclonal antibodies have indicated that TPO is related to the 'microsomal antigen' (McAg) (Czarnocka et al., 1985; Portmann et al., 1985), a target antigen involved in the autoimmune destruction of the thyroid in patients with Hashimoto's
thyroiditis (Roitt
et
al., 1964).
© IRL Press Limited, Oxford, England
To clarify the relationship between the two proteins, we have cloned the human TPO and McAg cDNAs independently. Analysis of the polypeptide sequences encoded in both clones demonstrates unequivocally that TPO and McAg are one and the same protein. In addition, it reveals a striking homology between TPO and the leucocyte protein myeloperoxidase (MPO), and demonstrates in its structure the presence of domains related to both nuclear (EGF-precursor, C4b) and mitochondrial (cytochrome C oxidase I) gene modules.
Results and discussion Cloning of TPO and McAg cDNA. Identification of a segment containing an epitope involved in autoimmunity A Xgtl 1 cDNA library constructed from a normal human thyroid was screened with a rabbit polyclonal anti-porcine TPO antibody and with a serum from a patient with Hashimoto's thyroiditis presenting a high titer (1/105) of anti-microsome autoantibodies (see Table I footnote). A series of TPO clones were obtained which allowed determination of the complete primary structure of the protein from the 3027-bp of the cDNA sequence (Figure Table I. Identification of McAg cDNA clones Serum
Ab anti-McAg Cl (titer)
C2
HI
10-4 lo-, 10-3
+
+
+
4
+
4
4
4
-4-
+
+ + + +
4 4
4 4 -
-
4 -
+
+
4
4
4
1
-
-
-
H2 H3 H4 H5 H6 H7 H8
10-4
±
lo-,
-
10-4
C3 C4 C5 C6 C7 C8
+
-
±
4-
-
10-4
+
H9
10-4
-
+ + +
HIO HII
lo-,
-
+
+
-
10-4
+ +
-
-
lo-, lo-,
-
-
H12 H13 H14 H15 H16 H17 H18
-
-
-
-
+
lo-5
-
--
10-4
1
+
+ + + +
+
lo-, lo-, lo-, lo-,
4
+
4
+
+
+
+
-
H19
Normal Size (kb) C2 cross-hybridization
4
-4
+
_
+
+ + 4 + +
4
+ + +
4
4-
-
-
+
-
+
-
4 -
+
±4
+
+
+
+
+
-
-
4 +
-
+
4
-
CIO
-
-
+
10-4
4
+
C9
4
+
-
+
+
+++
4
+ _ _
+
+ +
-
+
4
± + ++
4
+
_ +
-
+
-
4-
-
- - - 4 - 0.25 0.27 0.5 0.5 ND 0.9 0.3
