FR2837492A1 - New human apical iodide transporter useful for screening of compounds able to modulate apical iodide transport in cells for treatment, prevention or diagnosis of dysfunctional iodide transport - Google Patents

Abstract

Isolated, purified protein (I) comprising a fully defined sequence of 610 amino acids (S2) or a sequence at least 70-99 % similar to (S2) over its entire length, where the sequence AAH 17691 of the NCBI database is specifically exclude, is new. Independent claims are also included for: (1) a peptide fragment (Ia) of (I) containing at least 7 amino acids (aa); (2) isolated nucleic acid (II) that encodes (I), and its sense and antisense complements; (3) probe or primer (III) containing at least 8 base pairs (bp) of (II) but excluding AI261687, AI433656, AI793005 and AW195079 of GenBank and BC017691 of NCBI; (4) a recombinant vector that contains (II) or (III); (5) a cell transformed with the vector of (4); (6) antibodies (Ab) directed against (I) or (Ia); (7) a transgenic non-human mammal that contains cells transformed with (II); (8) screening for compounds able to modulate apical iodide transport in cells; (9) detecting a mutation in the AIT (apical iodide transporter) gene; and (10) a kit for the screening and detection methods.

Description

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 The present invention relates to a novel carrier of iodine and its applications in the prevention, diagnosis and treatment of genetic or acquired diseases involving a malfunction of this carrier.

 Iodine is an essential constituent of thyroid hormones, which are involved in the regulation of metabolism and development, particularly of the central nervous system. The synthesis of thyroid hormones involves the concentration of iodine in the thyroid lumen (colloid). Mammals have developed very effective mechanisms to collect and concentrate this rare element. In the thyroid, iodine present in the bloodstream is first accumulated in the thyrocytes, through the basement membrane. then it diffuses into the colloid, through the apical membrane. An accumulation of iodine would also be observed in the lumen of the stomach, as well as in the lumen of the salivary and mammary glands; iodine is also transported to the bloodstream, through the duodenal and renal epithelium and placenta.

 The iodine transport by the thyrocytes is a two-step process involving protein-like transporters at both the basement membrane and the apical membrane of the cell: - The NIS protein is located in the basolateral membrane, in direct contact with the bloodstream and intervenes in the accumulation of iodine in thyrocytes (For a review, see De la Vieja et al., Physiological Reviews, 2000, 80, 1083-1105). The NIS (Na / I Symporter or Sodium Iodide Symporter) gene has been cloned, particularly in rats, mice and humans: it encodes a protein that is located in the basement membrane of epithelial cells of the thyroid gland. stomach, as well as salivary and mammary glands. and catalyzes the coupled transport of sodium and iodine (2 Na / 11). of blood flow into epithelial cells, using the incoming sodium gradient. This gene belongs to the SSS (Sodium Solute Symporter) family which includes proteins involved in the coupled transport of sodium and small molecules such as glucose, vitamins such as biotin and pantothenate (SMVT for Sodium Multivitamin Symporter) or nucleosides.

 - The pendrine has been proposed as a potential apical transporter. The pendrin located in the apical membrane is the product of a gene (PDS gene) associated with an autosomal recessive disease characterized by deafness

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 combined with thyroid goitre (Pendred syndrome). However, the role of the pendrine in the physiology of the thyroid is not clearly established; the phenotypic variations observed in patients with this syndrome. suggest the existence of another apical iodine transporter system (Scott et al., Nature genetics, 1999, 21, 440, 443) and knockout mice for the PDS gene have severe auditory deficit but none physiological alteration of the thyroid (Everett et al., Human Molecular Genetics, 2001, 10, 153-161).

 It follows from the above that the transporter involved in the transport of iodine through the basement membrane of epithelial cells of the thyroid and salivary glands has been identified (NIS protein) but that the identity of the proteins involved in the transport of iodine through the apical membrane of the epithelial cells of the thyroid, stomach, salivary and mammary glands and through the apical and baso-lateral membranes of epithelial cells of the duodenum. kidney and placenta is unknown or only speculative.

 The inventors have identified a novel iodine transporter which is involved in the transport of iodine through the apical membrane of thyrocytes and in the accumulation of iodine in the colloid. This transporter, of a protein nature, is called AIT for Apical lodide Transporter or AIT proton; it is expressed mainly in the thyroid, but also in the salivary glands (submaxillary and parotid glands). the small intestine. the kidney, the adrenal glands and the prostate and to a lesser extent in the stomach, skin, lungs and testes.

 Such a transporter is useful in the prevention, diagnosis and treatment of genetic or acquired diseases involving dysfunction of the apical transport of iodine. It is also useful in nuclear medicine to control accumulation and prevent contamination by radioactive iodine.

 The present invention a. Consequently. for object an isolated and purified protein, characterized in that it has a sequence selected from the group consisting of: the sequence SEQ ID NO: 2 which corresponds to the human AIT protein (hAIT). and

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 sequences having, in their entirety, at least 70% identity or at least 85% similarity. preferably at least 80% identity or at least 90% similarity. and preferably at least 95% identity or at least 99% similarity to the sequence SEQ ID NO: 2, excluding the sequence having the accession number AAH17691 in the NCBI database.

 This protein represents a new apical iodine transporter and will be hereinafter referred to as AIT protein; the gene coding for this protein is hereinafter referred to as the AIT gene.

 The proteins according to the invention include any protein (natural, synthetic, semisynthetic or recombinant) of any prokaryotic or eukaryotic organism, in particular a human or non-human mammal, comprising or consisting of an acid sequence amines of a functional AIT protein.

 According to an advantageous embodiment of the invention, said protein is a mammalian protein.

 By "functional" is meant a protein having a normal biological activity. in this case an activity of apical iodine transporter. This protein may comprise silent mutations inducing no substantial change in its activity and producing no phenotypic changes.

 A functional AIT protein has the following properties: it has a sequence of about 610 amino acids and has a structure comprising: (i) 13 transmembrane domains having a helical structure; helices 1 and II each have a negatively charged residue (Aspl4 and Glu77) which can play a catalytic role, (ii) 3 potential sites of N-glycosylation (Asn 260, 481 and 485) and (iv) consensus sites of phosphorylation by a dependent cAMP kinase (RRMT between positions 50 and 53) or dependent Ca ++ [TSK (41-43). SCK (269-271). SER (377-379). SYK (576-578) and SGK (602-604)]; the positions of the amino acid residues being indicated with reference to the sequence of the hAIT protein (SEQ ID NO: 2), it is expressed mainly in the apical membrane of the thyrocytes but also in the epithelial cells of the salivary glands (sub-

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 maxillary and parotid). the small intestine. the kidney. the adrenal glands and the prostate and to a lesser extent in the stomach, the skin. the lung and the testicles.

 it is involved in the transport of iodine through the apical membrane of thyrocytes.

 The identity of a sequence with respect to a reference sequence is judged according to the percentage of amino acid residues that are identical, when the two sequences are aligned, so as to obtain the maximum of correspondence between them.

 A protein having an amino acid sequence having at least X% identity with a reference sequence is defined in the present invention as a protein whose sequence may include up to 100-X alterations per 100 amino acids of the sequence reference, while maintaining the functional properties of said reference protein, in this case its apical transport activity of iodine. For the purpose of the present invention, the term alteration includes deletions, substitutions or consecutive or dispersed insertions of amino acids in the reference sequence. This definition applies, by analogy, to nucleic acid molecules.

 The similarity of a sequence with respect to a reference sequence is judged according to the percentage of amino acid residues that are identical or different by conservative substitutions, when the two sequences are aligned in order to obtain the maximum of correspondence between them. For the purposes of the present invention, the term "conservative substitution" means the substitution of one amino acid for another that has similar chemical properties (size, charge or polarity), which generally does not modify the functional properties of the protein, the occurrence its activity of apical transport of iodine.

 A protein having an amino acid sequence having at least X% similarity to a reference sequence is defined in the present invention as a protein whose sequence may include up to 100-X non-conservative alterations per 100 amino acids of the reference sequence. For the purposes of the present invention, the term non-conservative alterations includes deletions, non-conservative substitutions or consecutive or dispersed insertions of amino acids in the reference sequence.

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 For example, among the known protein sequences. no identical or similar sequence was found: - the hNIS protein has 46% identity and 70% similarity with the hAIT protein, and - the hSMVT protein has 42% identity and 64% similarity with the protein Hait.

 The present invention also relates to a peptide, characterized in that it consists of a fragment of at least 7 amino acids of the protein AIT, as defined above.

 According to an advantageous embodiment of the invention, said peptide is constituted by a C-terminal fragment of said AIT protein. It is particularly useful for the production of antibodies specifically recognizing AIT protein.

 The present invention also relates to antibodies, characterized in that they are directed against the AIT protein or a fragment thereof, as defined above.

 According to an advantageous embodiment of the invention, said antibodies are directed against a C-terminal fragment of said AIT protein.

 According to the invention, said antibodies are either monoclonal antibodies or polyclonal antibodies.

 These antibodies can be obtained by conventional methods, known per se, including in particular the immunization of an animal with a protein or a peptide according to the invention, in order to make it produce antibodies directed against said protein or said peptide.

 Such antibodies make it possible in particular to determine the expression profile of the AIT protein and a possible alteration of this profile in a tissue or a biological sample, and consequently to diagnose a genetic or acquired disease involving a malfunction of this transporter.

 The present intention also relates to an isolated nucleic acid molecule (cDNA or genomic DNA fragment), characterized in that it has a sequence selected from the group consisting of: the sequences coding for an AIT protein such that defined above, and

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 the complementary sequences of the preceding ones, meaning or antisense.

 The invention encompasses sequences of alleles of the AIT gene derived from any mammal. as well as the sequences of the natural or artificial mutants of the AIT gene coding for a functional AIT protein as defined above. According to an advantageous embodiment of the invention. said sequence coding for an AIT protein is selected from the group consisting of: a) the sequence SEQ ID NO: 1 which corresponds to the cDNA of the hAIT protein, which sequence of 2509 bp comprises 5 'and 3' non-ends translated from 364 bp and 311 bp respectively, flanking an open reading phase of 1830 nucleotides, and b) the sequence between positions 2316362 and 2369604 of the sequence with the accession number NT009743.8 in the NCBI database , which sequence which corresponds to the hAIT gene, is located in the chromosomal region 12q23.1 and has an exon-intron organization comprising 15 exons (Table I). similar to that of the NIS gene.

Table 1: Exon-intron organization of the HAIT gene

<Tb>
<tb> Exon <SEP> n <SEP> Size <SEP> of <SEP> End <SEP> 3 '<SEP> of <SEP> End <SEP>5'<SEP> of <SEP> Size <SEP> of <SEP> End <SEP> 3 '<SEP> of <SEP> End <SEP>5'<SEP> of
<tb> the exon <SEP> (pb) <SEP> the exon <SEP> the intron <SEP> the intron <SEP> (pb) <SEP> the intron <SEP> the exon
<tb> (SEQ <SEP> ID <SEP> NO: <SE> (SEQ <SEP> ID <SEP> NO: <SEP> GATTCG
<tb> 15 <SEP> to <SEP> 29) <SEP> 30 <SEP> to <SEP> 43) <SEP>
<tb> 1 <SEP> 714 <SEP> TACGAG <SEP> gtaatttgca <SEP> 4936 <SEP> tttgcaacag <SEP> TATTTA
<tb> 2 <SEP> 66 <SEP> CAAACA <SEP> gtaagtagct <SEP> 2285 <SEP> tcccccctag <SEP> ATTCTG
<tb> 3 <SEP> 52 <SEP> ATCAAG <SEP> gtacatttta <SEP> 7002 <SEP> ttttctacag <SEP> TCACAG
<tb> 4- <SEP> 68 <SEP> ACACTG <SEP> gtacgtccag <SEP> 1315 <SEP> cccctcaaag <SEP> GGTGGT
<tb> 5 <SEP> 155 <SEP> CTGGAA <SEP> gtaagtgtct <SEP> 3013 <SEP> tgtcatttag <SEP> TTTTAA
<tb> 6 <SEP> 141 <SEQ> AAAACT <SEP> gtaagtcaca <SEP> 2950 <SEP> acttctttag <SEP> GTCTCT
<tb> 7 <SEP> 130 <SEP> GACCAG <SEP> gttcagtacc <SEP> 3166 <SEP> aatttttcag <SEP> CTCATG
<tb> 8 <SEP> 89 <SEP> ATTAAG <SEP> gtgtgaacta <SEP> 1220 <SEP> tcccttcag <SEP> CACAGT
<tb> 9 <SEP> 113 <SEP> GAATGA <SEP> gtaagtttct <SEP> 2706 <SEP> gtccatttag <SEP> GTGGG
<tb> 10 <SEP> 68 <SEP> TTGCAG <SEP> gtgagagctg <SEP> 11842 <SEP> atttttgtag <SEP> GCAGCA
<tb> 11 <SEP> 87 <SEP> TCAATT <SEP> gtaagtacaa <SEP> 1396 <SEP> tgtttacag <SEP> GGAGCA
<tb> 12 <SEP> 206 <SEP> TCAAAG <SEP> gtattgaatt <SEP> 4426 <SEP> gctctcttag <SEP> GACTCC
<tb> 13 <SEP> 104 <SEP> CAACAG <SEP> gtaactatct <SEP> 3652 <SEP> ttttttccag <SEP> GAGGAA
<tb> 14 <SEP> 80 <SEP> AAGAAA <SEP> gtgagttggc <SEP> 745 <SEP> tttcttttag <SEP> AAGAAG
<tb> 15 <SEP> 409 <SEP> ATACAC <SEP> acatatgcac
<Tb>

The nucleic acid moieties as defined above may be used as probes to isolate the AIT gene from other species or species.

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 alleles of this gene. in particular by screening a genomic DNA library or cDNA.

 The subject of the invention is also fragments of at least 8 bp of the nucleic acid molecules as defined above. excluding TSEs with accession numbers AI261687, AI433656. AI793005 and AW195079 in the GENBANK database and cDNA fragment having accession number BCO 17691 in the NCBI database. Such fragments may in particular be used as probes or as primers for detecting / amplifying nucleic acid molecules (mRNA or genomic DNA encoding an AIT protein as defined above.

 The nucleic acid molecules according to the invention are obtained by conventional methods, known per se, following the standard protocols such as those described in Cement Protocols in Molecular Biology (Frederick M.

AUSUBEL, 2000, Wiley and his Inc, Library of Congress. USA). For example, they can be obtained by amplification of a nucleic sequence by PCR or RTPCR, by screening genomic DNA libraries by hybridization with a homologous probe. or by total or partial chemical synthesis.

 These different nucleic acid molecules make it possible either to determine the transcription profile of the AIT gene and a possible alteration of this profile in a tissue or a biological sample, or to highlight the AIT gene. allelic variants of this gene and a possible functional alteration of this AIT gene (substantial change in the apical iodine transporter activity of the AIT protein) resulting from a mutation (insertion, deletion or substitution) of one or more nucleotides at at least one exon of said gene. Such mutations include, in particular, deletions, insertions or nonconservative substitutions at codons corresponding to amino acid residues located in a domain essential for the biological activity of AIT protein.

 The transcripts of the AIT gene are, for example, amplified by RT-PCR using primers selected from the group consisting of the sequences SEQ ID NO: 3 to 13, and

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Exons 1 to 15 are amplified by PCR using 15α primers
50 bp located in the introns flanking said exons, at a distance of about 20 to
100 bp upstream and / or downstream of said exons.

 Consequently. these different nucleic acid molecules make it possible to diagnose a pathological state or a genetic disease involving a dysfunction of this transporter and to screen for substances capable of modulating (activating or inhibiting) the transcription of the AIT gene.

 The present invention also relates to a recombinant vector, characterized in that it comprises an insert selected from the group consisting of nucleic acid molecules encoding a protein AIT and fragments thereof as defined above.

 Preferably, said recombinant vector is an expression vector wherein said nucleic acid molecule or a fragment thereof is under the control of appropriate transcriptional and translational regulatory elements.

 These vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se. Many vectors where a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell. are known in themselves; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or integration into the chromosomal material of the host ), as well as the nature of the host cell.

 The subject of the present invention is also prokaryotic or eukaryotic cells transformed by a recombinant vector as defined above.

 The recombinant vectors and the transformed cells as defined above are particularly useful for the production of AIT proteins and peptides according to the invention or in substitutive gene therapy.

 The present invention also relates to a non-human transgenic mammal, characterized in that it contains cells transformed with at least one nucleic acid molecule as defined above.

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The non-human transgenic mammals and the transformed cells as defined above, are useful in particular for screening drugs capable of modulating the activity of the apical iodine transporter of the protein.
AIT. as defined above.

 The present invention also relates to a medicament for the treatment of diseases involving a malfunction of the apical transport of iodine, characterized in that it includes: a nucleic acid molecule, coding for a protein AIT as defined herein above, a recombinant vector as defined above, an AIT protein as defined above or an antibody as defined above.

 The present invention also relates to a reagent for detecting a disease involving a dysfunction of the apical transport of iodine, characterized in that it is selected from the group consisting of: nucleic acid molecules and their fragments such as as defined above (probes, primers) and the antibodies as defined above.

 The present invention also relates to a method for screening a substance capable of modulating (activating or inhibiting) the apical transport of iodine in cells, characterized in that it comprises: the preparation of cells expressing a protein AIT as defined above.

 contacting said cells with a substance to be tested, measuring by any appropriate means the effect of said substance on the activity of said AIT protein, and selecting substances capable of modulating said activity.

 For the purposes of the present invention, the term "activity of the AIT protein" means both the expression of the AIT protein or corresponding transcripts (mRNA), and the biological activity of said protease AIT. in this case the activity of apical iodine transporter.

 Said measurement is carried out by standard mRNA, protein or iodine influx techniques which are known per se; by way of non-limiting example, mention may be made of the following techniques: RT-PCR,

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 Northern-blot, Western-blot. RIA, ELISA, immunoprecipitation, incorporation of radiolabelled iodine.

 Advantageously. said measurement is carried out using the detection reagents as defined above (antibodies, probes, primers).

 Substances capable of inhibiting the apical transport of iodine in cells have applications for the treatment of pathologies involving hypersecretion of thyroid hormones, as well as for the prevention and treatment of contaminations by radioactive iodine. Indeed, a selective inhibition of the activity of the protein AIT in the tissues accumulating the iodine as the thyroid could limit the risks of tumorization of these tissues and the protein AIT which can be involved in the re-assimilation towards the blood, of the iodine passed in the primary urine, an inhibition of the activity of the protein AIT would also increase the elimination, in the urine, of the radioactive iodine assimilated accidentally.

 The present invention also relates to a method for detecting a mutation in the AIT gene, characterized in that it comprises: - the preparation of a sample of nucleic acids, - the detection of the presence, in said sample of nucleic acids, a mutation in the sequence coding for an AIT protein as defined above, using at least one probe or a primer as defined above.

 According to an advantageous embodiment of said detection method, said mutation is located at an exon of the AIT gene.

 Said detection is carried out by the conventional techniques which are known per se, for example: (i) by amplification of a region of said AIT gene likely to contain a mutation, then detection of said mutation by sequencing or by digestion with an enzyme of appropriate restriction of the PCR product obtained, or (ii) by hybridization with a labeled probe specific for a region of said AIT gene likely to contain a mutation, then direct detection of mismatches and / or digestion with an appropriate restriction enzyme.

 The invention further relates to a detection and / or screening kit for the implementation of the methods as defined above, characterized in that it includes at least one reagent as defined above.

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11
In addition to the foregoing, the invention further comprises other arrangements which will become apparent from the following description, which refers to examples of implementation of the AIT protein of the present invention as well as to the accompanying drawings in FIG. which: - Figure 1 illustrates the comparison of the sequences of hAIT proteins (SEQ ID NO: 2). hNIS (GENBANK No. U66088) and hSVMT (GENBANK No. AF69307) identical amino acids are represented by a line. The 13 potential transmembrane domains are boxed and the residues of these negatively charged domains (-) or positively (+) are indicated. Potential sites of glycosylation (g), phosphorylation by: a cAMP dependent protein kinase (pa), a protein kinase C (pc) or a tyrosine kinase (ptyr), are also indicated.

 FIG. 2 illustrates the quantitative RT-PCR analysis of the hAIT mRNA expression profile in different human tissues: the results are normalized using a probe corresponding to the 18S ribosomal RNA. The results are expressed in arbitrary units, relative to the expression of mRNA in the thyroid (100%).

 FIG. 3 illustrates the immunohistochemical detection of the hAIT protein in the normal thyroid (FIG. 3A) and in the maxillary gland (FIG. 3B). magnification x 250. Intense immunostaining of the apical membrane of thyrocytes and ductal cells of the submaxillary gland is observed with anti-peptide hAIT serum (577-610).

 FIG. 4 illustrates the analysis, by incorporation of radiolabeled iodine (125I), of the iodine influx in HEK-293-T and COS-7 cells transiently transfected with an expression plasmid of the hAIT protein, alone or in combination with a plasmid expressing the mNIS protein. Each measurement is done in triplicate and the results, corresponding to the mean + s. e.m, are expressed in thousands of cpm / well.

 FIG. 5 illustrates the kinetic analysis, by incorporation of radiolabelled iodine (125I), of the iodine influx into CHO cells stably transfected with an hAIT protein expression plasmid (hAIT) or non-transfected (control). The results, corresponding to the mean ~ s.e.m, are expressed in

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 number of cpm / well. FIG. 5A illustrates the influx of iodine measured under the conditions as described in Example 1: transfected cells treated with perchlorate (-O-) or non-treated (-), control cells treated (- # - ) or untreated (- # -). The measurements are made in triplicate, at different times, between 0 and 15 min, after the addition of 125I. FIG. 5B illustrates the influx of iodine in the presence of a counter-flux, measured under the conditions as described in Example 1: transfected cells (- # -), control cells (- # -). The measurements are made in triplicate, at different times, between 0 and 6 min, after the addition of 125I.

séquences est réalisée à l'aide du programme BLAST (http:..'7w ww.ncbi.nlm.nih.gov/). Les sites potentiels de glycosylation et de phosphorylation ont été localisés à l'aide du

programme Pro.site fourni par EXPASY (http:7\vww.expasv~~ch/prosite). EXAMPLE 1 AND METHODS 1) Equipment
Reagents are provided by SIGMA-ALDRICH CHEMICHAL. Restriction endonucleases and T4 DNA ligase are provided by BIOLABS. The cloning kit (TA cloning kit) is provided by PROMEGA. E. coli strain XL2-Blue bacteria are provided by STRATAGENE. The sequence of the oligonucleotide primers is determined using the program available on the website (htttp: //alces.med.umn.edu/rawprimer.html) and the oligonucleotide primers are synthesized by SIGMA-GENOSYS. The analysis of

sequences is performed using the BLAST program (http: .. '7w ww.ncbi.nlm.nih.gov/). Potential sites of glycosylation and phosphorylation were localized using the

Pro.site program provided by EXPASY (http: 7 \ vww.expasv ~~ ch / prosite).

 Normal tissue samples were removed from the tumor site; during surgical procedures. The samples were frozen at -70 ° C. in isopentane and then stored in liquid nitrogen.

2) Methods a) cloning the cDNA of the hAIT protein by RT-PCR
Human kidney cDNAs (Marathon ready cDNAs, CLONTECH) were used as the template. PCR reactions are performed in a Robocycler (STRATAGENE). using the high fidelity PCR system (ROCHE) and possibly Hotstart tubes (MOLECULAR BIO-PRODUCTS). according to the manufacturer's instructions. The PCR amplification is carried out under the following conditions: an initial denaturation step at 94 ° C. for 3 min is followed by 35 to 40 cycles alternating a denaturation step at 94 ° C.

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for 50 s, a hybridization step at 50 C for 50 s and a step of elongation at
C. for 1 to 3 minutes and a final elongation step is carried out at 69 ° C. for 6 to 8 minutes. The PCR amplification products are separated by agarose gel electrophoresis, purified and cloned into the plasmid pGEM-T (PROMEGA). The ends of the cDNAs are amplified by the Rapid Amplification of cDNA (RACE) technique.
Ends), using a commercial kit (Marathon kit, CLONTECH). The cloned fragments are sequenced by GENOME EXPRESS.

 In a first step. cDNA fragments covering the entire hAIT sequence were amplified, cloned into plasmid pGEM-T, and sequenced. Namely: a 350 bp cDNA fragment corresponding to part of the 5 'non-coding region and at the 5' end of the open reading phase of the hAIT protein was amplified using the pair of primers SEQ ID NO: 3 and SEQ ID NO: 4.

a fragment of about 1682 bp. located between positions 273 and
1954 of the hAIT cDNA was amplified in two steps using: a first pair of primers (sense primer SEQ ID NO: 5, located between positions 273 and 293 and SEQ ID antisense primer NO: 6, located between positions 1261 and 1283) and a second pair of primers (sense primer SEQ ID NO: 7, located between positions 694 and 719 and antisense primer SEQ ID NO: 8, located between positions 1929 and 1954), and the 3 'end of the hAIT cDNA was amplified by the RACE technique, using as a specific primer. the sense primer SEQ ID NO: 9, located between positions 1630 to 1652 of the hAIT cDNA.

 In a second step, a fragment containing the entire hAIT coding sequence was amplified by PCR using the primer pair: SEQ ID NO: 10: sense primer 5'-AAATCTAGAGCCACCnucleotides of positions 365 to 388 hAIT-3 'cDNA, comprising a NotI site and a Kozak sequence. placed immediately before the initiation codon of the hAIT open reading phase. and SEQ ID NO: 11: 5'-TTAGAATTC-nucleotide antisense primer from positions 2187 to 2209 of the hAIT cDNA, comprising an EcoRI site located just upstream of the stop codon.

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The amplification product obtained was inserted between the NotI sites and
EcoRI of the plasmid pcDNA3.1 (INVITROGEN). b) Determination of the expression level of the hAIT gene by RT-PCR in real time
The distribution of mRNAs in different tissues is analyzed by a real-time quantitative RT-PCR method. according to the Taqman technique (PERKIN-
ELMER APPLIED BIOSYSTEMS).

 Total RNAs are extracted from different tissues, using a commercial DNA and RNA extraction kit, following the manufacturer's instructions (QIAGEN). The quality of the RNA is verified by agarose gel electrophoresis. 1 g of total RNA is retro-transcribed using random hexameric primers (PERKINELMER APPLIED BIOSYSTEMS), following the protocol described in Lazar et al., J. Clin. Endocrinol. Metab., 1999, 84, 3228-3231. The cDNAs thus obtained are diluted by a factor of 20 in nuclease-free water (PROMEGA).

Oligonucleotide primers (SEQ ID NO: 12 and SEQ ID NO:
13) and the Taqman probe (SEQ ID NO: 14) were selected in regions overlapping introns of the hAIT gene.

The quantitative real-time PCR reactions were performed in a volume of 50 l containing a quantity of cDNA corresponding to 20 ng of total DNA, using the commercial kit (Taqman PCR core reagent kit), according to the instructions. from the manufacturer (PERKIN-ELMER APPLIEL) BIOSYSTEMS). To normalize the differences between the amounts of RNA added in the reactions, the 18S ribosomal RNA was amplified simultaneously as an internal control (Lazar et al., Supra). Expression of hAIT in the different tissues is expressed, relative to a reference sample, consisting of a mixture of total normal thyroid RNAs. Each experimental point is duplicated and the coefficient of variation in the same experiment is less than 1%. c) Synthesis of peptides
A peptide located between amino acids 577 and 610 of the hAIT protein was synthesized according to conventional solid phase synthesis techniques, using an automatic synthesizer (model 431 A, PE APPLIED BIOSYSTEMS).

The identity and purity of the synthesized peptide was verified by: (i) amino acid analysis by an automatic analyzer (LKB), (ii) chromatography

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fi ) tr.é;l.l)..f.t.i.Q.mJXm1$.i.9.r.. high performance liquid phase (HPLC), and (iii) microsequencing of each peak obtained in HPLC, using an automatic sequencer (model 477A.
APPLIED BIOSYSTEMS). The hAIT peptide was conjugated at the residue level
Tyr578, to KLH protein (Keyhole Limpet Hemocyanine), using benzedine as a coupling agent. d) Production and characterization of anti-peptide hAIT antibodies
Two rabbits were immunized by intradermal injection of hAIT peptide coupled to KLH. according to a protocol comprising a first injection, followed by three booster injections, spaced apart by an interval of 3 weeks. Rabbit blood was then collected and the titer of anti-hAIT antibody sera determined by ELISA. using microtiter plates coated with hAIT peptide and anti-rabbit immunoglobulin antibodies coupled to peroxidase (NORDIC). e) Immunohistochemistry
Serial sections spaced 5 mm apart. Normal thyroid samples were taken using a cryostat, and the sections were fixed in acetone for 10 minutes. The sections were then incubated for 1 hour with anti-peptide hAIT serum diluted by a factor of 50. After three successive 5 min washes in 15 mM Tris-HCl buffer pH 7.4, the sections were incubated with an antibody. anti-rabbit immunoglobulin coupled to biotin (K674, DAKO). After three successive washings of 5 min in the same Tris-HCl buffer. the reaction is revealed in the presence of the fast red substrate (K699, DAKO). The negative controls consist of sections incubated with a pre-immune serum or a pre-adsorbed hAIT anti-peptide serum in the presence of an excess of hAIT peptide. f) Analysis of 125I incorporation by HEK-293T cells. COS-7 and CHO transfected

fi) tr.é; ll) .. ftiQmJXm1 $ .i.9.r ..

 HEK-293-T cell lines (human embryonic kidney cells stably transfected with SV40 T antigen, EACCC number 85120602) and COS-7 (African green monkey kidney cells EACC number 87021302) are seeded. in 12-well plates (coated with polylysine, in the case of HEK-293-T cells). and cultured at 37 C in the presence of CO2, in medium

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 DMEM (GIBCO) containing 2 mM glutamine (GIBCO) and 10% fetal calf serum (GIBCO). When the cells reach 70% confluency, they are transiently transfected with different plasmids. using the FugeneR reagent (ROCHE): the recombinant pcDNA3.1 plasmid containing the hAIT cDNA (pcDNAhAIT). the recombinant pcDNA3.1 plasmid containing the mouse NIS protein cDNA (pcDNA-mNIS Perron et al., J. Endocrinol., 170. 185-196), the plasmid pcDNA-hAIT in combination with the plasmid pcDNA- mNIS or the plasmid pcDNA3.1vide.

 The expression of the hAIT protein by the transfected cells was verified by immunohistochemistry. using anti-peptide hAIT serum.

 3 days after transfection. the cells are rinsed twice with HBSS solution buffered with 10 mM Hepes pH 7.0 (GIBCO). The influx of iodine is analyzed by incorporation of 125 I by the cells, according to the protocol described in Weiss et al., Endocrinology. 1984, 114, 1090-1098. More specifically, a buffered HBSS solution (HBSS buffer) containing 50 M Na125I (50Ci / mol) is added to each well (in triplicate) and the cells are incubated at 37 ° C. for 45 min.

Then, the radioactive solution is removed and the cells are rinsed rapidly, 3 times, with a cold HBSS solution. Finally, the cells are permeabilized with ethanol and the 125 I release is measured using a liquid scintillation counter.

12) transfectipns.stables f2) WJ.p.ft; j9D ~~ tR! -
CHO cells (Chinese Hamster Ovary, EACC No. 85050302) are inoculated in F12 medium (GIBCO) containing 2 mM glutamine and 10% fetal calf serum and incubated at @ 7 C in the presence of CO 2, they reach 70% confluence. The cells are then transfected with the plasmid pcDNA-hAIT in the presence of the FUGENER reagent (ROCHE) and then selected in the presence of G418 (500 μg / ml, INVITROGEN). The expression of the hAIT protein by the G418-resistant clones was verified by immunohistochemistry using anti-peptide hAIT antiserum. The iodine influx is analyzed by incorporation of 125I as described above, with the following exceptions: (1) the cells are incubated in the presence of 125I, for a variable duration between 0 and 15min and (2) 1 mM perchlorate is optionally added to the buffer

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HBSS containing radioactive iodine. The influx of iodine is also measured in the presence of a counter-flow: the cells are washed twice with HBSS buffer, incubated for 30 min in HBSS buffer containing 1 mM NaI, then HBSS buffer containing 50 M Na125I (50Ci / mol) is added to each well (in triplicate) and the cells are incubated at 37 ° C. for a variable duration of between 0 and 6 min.

 EXAMPLE 2 Cloning, Sequencing and Analysis of the cDNA of the hAIT Protein

 Sequence analysis, cloning and sequencing of the hAIT cDNA were performed according to the protocols described in Example 1.

Four human cDNA fragments with strong homology to the cDNA sequence of the NIS protein were identified in the NCBI EST database (AI261687, AI433656, AI793005 and AW195079). Of these fragments, three have overlapping sequences (AI261687, AI433656,
AI793005) corresponding to the 5 'untranslated end and part of an open reading phase; the longest protein (50 amino acids), which is derived from the AI261687 fragment, has 46% identity and 78% similarity to the N-terminal sequence of the hNIS protein (GENBANK # U66088). The AW195079 fragment contains an open reading phase encoding a 117 amino acid fragment having 47% identity and 83% similarity to residues between positions 68 and 184 of the hNIS protein.

 RT-PCR reactions carried out using the primers SEQ ID NO: 3 (sense primer derived from the overlapping sequences AI261687, AI433656 and AI793005) and the primer SEQ ID NO: 4 (antisense primer derived from the sequence AW195079) and human kidney cDNA can amplify a product of 350 bp; these results show that the 4 ESTs come from the same mRNA.

 The sequences of the 5 'and 3' ends of this new messenger were determined by the RACE technique and a cDNA corresponding to the complete sequence of this mRNA was cloned and sequenced. The results show that this cDNA consists of a sequence of 2509 bp (SEQ ID NO: 1), comprising untranslated 5 'and 3' ends of respectively 364 bp and 311 bp, flanking an open reading phase of 1830 nucleotides.

 This open reading phase encodes a protein of 610 amino acids (SEQ ID NO: 2). with 46% identity and 70% similarity to the

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hNIS protein and 42% identity and 64% similarity to the hSMVT protein (FIG.
1).

The potential structure of this protein called hAIT for human Apical Iodide Tram: carry, obtained from the model of the NIS protein reviewed by Levy et al., J. Biol. Chem., 1998, 273. 22657-22663 is shown in Figure 1. The hAIT protein comprises 13 potential transmembrane domains having a helical structure. Helices 1 and II each have a negatively charged residue (Aspl4 and Glu77) which can play a catalytic role; these residues are conserved at equivalent positions in the NIS protein (Dai et al., Nature, 1996,
379, 458-460). It also includes 3 potential N-glycosylation sites (Asn 260,481 and 485); these sites are also present at equivalent positions in the NIS protein. It further has consensus phosphorylation sites by a dependent cAMP kinase (RRMT located between positions 50 and 53) or Ca ++ dependent [TSK (41-43). SCK (269-271). SER (377-379), SYK (576-578) and SGK (602-604)].

EXAMPLE 3 Location and organization of the hAIT gene

The hAIT gene was located in the chromosomal region 12q23.1. The intron-exon organization of this gene shows that the coding sequence of hAIT is interrupted by 14 introns. The sequence of each intron-exon junction is shown in Table I.

 The organization of the hAIT gene is very similar to that reported for the NIS gene (Smanik et al., Endocrinology, 1997, 138, 3555-3558) which shows the presence of 14 introns located at positions equivalent to those of the hAIT gene.

EXAMPLE 4 Analysis of the Level of Expression of the HAIT Gene in Human Tissues
The expression of the hAIT gene was analyzed by quantitative RT-PCR, according to the protocol described in Example 1. The results show that the highest expression is detected in the normal thyroid (FIG. 2). In the digestive and related tissues, the expression of hIA Test detected in the submaxillary glands and, to a lesser extent. in the parotid gland and the small intestine. A very weak signal has also been detected in the gastric mucosa, while other tissues including the # esophagus. the colon. the pancreas and the liver do not express the HAIT gene. In the kidney, adrenal gland and prostate a significant expression of hAIT mRNA is

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detected. at levels 10 to 50 times higher than those seen in the thyroid. HAIT transcripts are also detected in the lung, skin and testis at a single level.
100 to 500 times lower than that observed in the thyroid, no expression of the hAIT gene was detected in the muscle, the heart, the ovary, the breast and the endometrium.

 EXAMPLE 5 Location of the hAIT protein in the thyroid and submaxillary salivary gland.

 The localization of the hAIT protein in the thyroid and in the submaxillary salivary gland was analyzed by immunohistochemistry, according to the protocol described in Example 1. The results presented in FIG. 3 show that the hAIT protein is localized at the level of the apical membrane of thyrocytes (Figure 3 A). A pattern of heterogeneous expression is observed with greater immunostaining of certain follicles and certain regions of the same follicle, the majority of thyrocytes are labeled but a lack of labeling is however observed in some thyrocytes. In the salivary gland, only the ductal cells are labeled with antiserum (Figure 3B). the signal is diffusely distributed throughout the cytoplasm.

EXAMPLE 6 Function of the hAIT Protein in Mammalian Cells
The iodine flux in the cells expressing the hAIT protein was analyzed by transient transfection of HEK-293-T cells or COS-7 cells or by stable transfection of CHO cells, according to the protocol described in Example 1.

 Immunohistochemistry analysis of the expression of the hAIT and mNIS proteins in the transiently transfected cells using hAIT anti-peptide serum and an antibody against the rat NIS protein respectively (Boland et al. Cancer Res., 2000, 60, 3484-3492). shows that maximal expression is observed 48 h and 72 h after transfection, respectively for mNIS and hAIT. These results indicate a difference in synthetic kinetics or cellular transport between NIS and AIT proteins.

 Reactive iodine incorporation results in the transiently transfected cells shown in Figure 4 show that, compared to cells expressing the mNIS protein alone, which accumulate iodine, a decrease in this equilibrium is observed. of iodine accumulation in the

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 cells co-expressing the proteins the hAIT protein and mNIS. These results indicate that the hAIT protein catalyzes the diffusion of iodine through the cell membrane. Because of the location of the hAIT protein in the apical membrane of thyrocytes (Example 5). these results indicate that the hAIT protein catalyzes the facilitated diffusion of iodine through the apical membrane of thyrocytes and the transfer of iodine from thyrocytes to the colloid. In contrast, NIS protein, which is expressed at the level of the basement membrane of thyrocytes, in contact with the blood circulation, catalyzes the transport of iodine through the basement membrane of thyrocytes and the accumulation of iodine in the cells. thyrocytes.

 The iodine incorporation results in the stably transfected cells shown in Figure 5 show a significant increase in the initial influx of iodine (Figure 5A), relative to the non-transfected cells. This increase is inhibited by a competitive inhibitor of iodine transport by NIS protein (perchlorate). This increase in the initial influx of iodine is also observed in the presence of a counter-flow (FIG. 5B). which indicates that the transfer of iodine is actually mediated by a carrier.

 These results indicate that hAIT protein catalyzes iodine transport across the cell membrane but not its intracellular accumulation.

As a result, AIT protein contributes to the facilitated transfer of iodine into the tissues in which it is expressed, including the thyroid, salivary glands, kidney, small intestine and stomach; in the thyroid, it catalyzes the transport of iodine in the colloid; through the apical membrane of thyrocytes.

 As is apparent from the foregoing, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (14)

1) isolated and purified protein, characterized in that it has a sequence selected from the group consisting of: - the sequence SEQ ID NO: 2, and - the sequences having, in their entirety, at least 70% identity or at least 85% similarity, preferably at least 80% identity or at least 90% similarity, and preferably at least 95% identity or at least 99% similarity to the sequence SEQ ID NO: 2, excluding the sequence having the accession number AAH17691 in the base of NCBI data.  2) Protein according to claim 1, characterized in that it is a mammalian protein.  3) Peptide, characterized in that it consists of a fragment of at least 7 amino acids of the protein according to claim 1 or claim 2.  4) Peptide according to claim 3, characterized in that it consists of a C-terminal fragment of said protein.  5) isolated nucleic acid molecule, characterized in that it has a sequence selected from the group consisting of: the sequences coding for a protein according to claim 1 or claim 2, and the complementary sequences of the preceding sequences, meaning or anti-sense.  6) isolated nucleic acid molecule according to claim 5, characterized in that it is selected from the group consisting of: a) the sequence SEQ ID NO: 1, and b) the sequence located between positions 2316362 and 2369604 of the sequence with accession number NT009743. 8 in the NCBI database.  7) probe or primer, characterized in that it consists of a fragment of at least 8 bp of the nucleic acid molecules according to claim 5 or claim 6, excluding sequences having the numbers of accession AI261687, AI433656, AI793005 and AW195079 in the GENBANK database and accession number BC017691 in the NCBI database. <Desc / Clms Page number 22>  - 8) recombinant vector, characterized in that it comprises an insert selected from the group consisting of the nucleic acid molecules according to claim 5 or claim 6 and their fragments as defined in claim 7.  9) Cells, characterized in that they are transformed by a recombinant vector according to claim 8.  10) Antibodies, characterized in that they are directed against the protein according to claim 1 or claim 2 or the peptide according to claim 3 or claim 4.  It ') A medicament for the treatment of diseases involving a malfunction of the apical transport of iodine, characterized in that it comprises a molecule selected from the group consisting of: protein according to claim 1 or claim 2, a peptide according to claim 3 or claim 4, a nucleic acid molecule according to claim 5 or claim 6, a recombinant vector according to claim 8 and an antibody according to claim 10.  12) A detection reagent for a disease involving a malfunction of the apical transport of iodine, characterized in that it is selected from the group consisting of: a nucleic acid molecule according to claim 5 or claim 6, a Probe or primer according to claim 7 and an antibody according to claim 10.  13) non-human transgenic mammal, characterized in that it contains cells transformed with at least one nucleic acid molecule according to claim 5 or claim 6.  14) A method of screening a substance capable of modulating the apical transport of iodine in cells, characterized in that it comprises: - the preparation of cells expressing a protein as defined in claim 1 or claim 2, - bringing said cells into contact with a substance to be tested, - measuring by any appropriate means the effect of said substance on the activity of said protein, and - the selection of substances capable of modulating said activity. <Desc / Clms Page number 23>  15) Method for detecting a mutation in the AIT gene, characterized in that it comprises: - the preparation of a sample of nucleic acids, - the detection of the presence, in said nucleic acid sample, of a mutation in the coding sequence for a protein according to claim 1 or claim 2, using at least one probe or primer according to claim 7.  16) Kit for detection and / or screening, characterized in that it includes at least one reagent according to claim 12.