Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Reexamination Certificate
1999-12-22
2003-09-16
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
C530S300000, C435S069100, C435S235100, C435S325000, C536S023500
Reexamination Certificate
active
06620914
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to identification and characterisation of Islet-Brain 1 (IB1), a transcriptional activator that is involved in the control of the GLUT2 and insulin genes by interacting with homologous cis-regulatory elements of the GLUT2 and insulin promoters, and to materials and methods deriving from this work. In particular, the present invention relates to the uses of IB1 nucleic acid, IB1 polypeptides, IB1 antagonists and antibodies in the diagnosis, and prophylactic and therapeutic treatment of conditions such as diabetes, neurological diseases such as dementia and/or parkinsonism, cancer and in the promotion or inhibition of apoptosis.
BACKGROUND OF THE INVENTION
The GLUT2 facilitated glucose transporter isoform is a membrane protein present in the pancreatic &bgr;-insulin-secreting cells, the basolateral membrane of intestinal and kidney absorptive cells, in hepatocytes and in a subset of neurons (21,31,44). In these cells, GLUT2 catalyzes the transepithelial transport of glucose. In pancreatic islets, GLUT2 allows a rapid equilibration of glucose between the extracellular space and the interior of the cells and it may play a crucial role in the glucose signalling mechanism leading to insulin secretion (43). However, the relative importance of GLUT2 in the sensing of the &bgr;-pancreatic cells to glucose remains debated. In human &bgr;-cells, the level of expression of GLUT2 is low and the intracellular glucokinase activity seems to be the rate-limiting step in the glycolytic pathway (5,11). On the other hand, insulinoma cells that had lost their normal glucose responsiveness have low GLUT2 content, but some glucose sensitivity may be recovered after reintroducing GLUT2 expression through stable transfection of these cells (10,16). Furthermore, transgenic mice that express GLUT2 antisense RNAs driven by the insulin promoter led to an 80% reduction in GLUT2 which was paralleled by a decreased glucose-induced insulin secretory response and by the onset of diabetes (48). These observations are critical since several experimental models of diabetes have shown that GLUT2 expression is dramatically reduced specifically in the pancreatic &bgr;-cells, and that this mechanism could participate to the onset of the disease (18,29,30,32,45-47). Therefore, while GLUT2 levels are unchanged or even upregulated in several tissues such as the liver and the intestine during the hyperglycemic conditions observed in diabetes, the same gene undergoes a drastic dysregulation only in the pancreatic islets.
A fragment of the murine GLUT2 promoter has been cloned and shown to be glucose-responsive when transfected into differentiated insulin-producing cells or into hepatocytes (35,36,52). Important cis-regulatory sequences were identified within this promoter region including a functionally responsive PDX-1 element, a cyclic AMP responsive element, and three cis-elements termed GTI, GTII and GTIII (3,36,53). The presence of GTI, II and III are both sufficient and necessary to confer pancreatic-specific expression to a reporter gene in vitro or in vivo, using a transgenic mice approach (3,51). GTI and GTIII have been previously shown to bind distinct, but ubiquitously expressed trans-acting factors.
SUMMARY OF THE INVENTION
The present invention is based on successful expression cloning of a transcription factor that binds to the GTII element of the GLUT2 and insulin genes from a differentiated insulin-secreting cDNA library. In part, the success of this exercise was based on the inventors' realisation of the importance of GTII and the library they constructed to find the IB1 gene. The IB1 polypeptides described herein are relatively large, and cloning them was achieved by the construction by the inventors of a high quality cDNA library for expression cloning.
This factor is abundantly expressed in the pancreatic islets and in the brain and has been named IB-1 for Islet-Brain 1. Both human and rat IB1 genes and polypeptides have been obtained. The rat IB1 cDNA (SEQ ID NO: 1) encodes a 714 amino acid protein (SEQ ID NO: 2) and the human IB1 cDNA (SEQ ID NO: 3) a 711 amino acid protein (SEQ ID NO: 4). The cDNAs encoding the rat and human IB1 polypeptides have a 94% sequence identity and the polypeptides have a 97% amino acid sequence identity (see the alignment of the sequences in FIG.
1
D), and have a proline-rich region and a putative basic helix-loop-helix domain (bHLH). The IB1 gene is highly expressed in the pancreatic islets and in the brain and to a much lesser extent in the heart and the kidney. In the Langerhans islets and in &bgr;-cell lines, these transcripts are translated into immunodetectable cytoplasmic and nuclear protein. When tested in vitro, IB1 bound specifically to the GTII cis element of the GLUT2 gene and to an homologous regulatory sequence of the insulin promoter termed RIPE3. This rat insulin promoter element 3 (RIPE3) is an important enhancer sequence sufficient to confer &bgr;-cell specific expression to the insulin gene. Functionally, IB1 transactivated the proximal region of the GLUT2 promoter linked to a luciferase reporter gene and was also a potent activator of the insulin gene. This effect is mediated through the RIPE3 sequence as demonstrated by the observation that multiple copies of this enhancer sequence cloned 5′ of an heterologous promoter was transactivated by an expression vector encoding IB1 in transient transfection studies. IB1 appears to function only in insulin-secreting cells as no transactivation was observed in non-pancreatic or in glucagon-producing cell lines. These data demonstrate the presence of a novel transcriptional activator abundantly expressed in the endocrine pancreas and which participates to the proper &bgr;-cell specific control of the GLUT2 and the insulin genes through homologous sequences present in both promoters.
The nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2) of rat IB1 are shown in FIG.
1
A. The human IB1 cDNA (SEQ ID NO: 3) is shown in
FIG. 1E
, with the translated amino acid sequence (SEQ ID NO: 4) shown in FIG.
1
F. The human IB1 gene is located on chromosome 11 at 11p11.12 on the LDB cytogenic map. The IB1 gene is adjacent to markers D11S134 and D11S3979.
The human cDNA was constructed as RNA using tissue obtained from a surgically removed human insulinoma. Poly A
+
RNA was extracted and a cDNA library constructed and subsequently screened with a radiolabelled rat IB1 cDNA probe. This allowed the inventors to isolate the human cDNA encoding IB1. This cDNA was then used as a probe to clone the human IB1 gene from a bacterial artificial chromosome (BAC). Several clones were obtained and part of them sequenced. The above protocol was then used to complete the sequencing of the human IB1 nucleic acid shown in
FIG. 1E
(SEQ ID NO: 3).
The human IB1 gene is multiexonic and is located in chromosome 11p11.12. The chromosomal mapping was obtained by FISH experiments and PCR of hybrid cells (hamster-human) using as a probe the multiexonic IB1 gene. IB1 is expressed in the brain of rat, mouse and human species and in tissues with a high degree of similarity such as the endocrine pancreas (many neuronal features are present in the insulin &bgr;-cells).
In a first aspect, the present invention provides a substance which is an isolated polypeptide comprising a polypeptide having the amino acid sequence set out in
FIG. 1A
(SEQ ID NO: 2) or
FIG. 1F
(SEQ ID NO: 4).
In a further aspect, the present invention provides a substance which is an isolated polypeptide having greater than 80% amino acid sequence identity with the amino acid sequence set out in
FIG. 1A
(SEQ ID NO: 2) or
1
F (SEQ ID NO: 4).
In a further aspect, the present invention provides a substance which is a polypeptide which is a mutant, variant, derivative or allele of any one of the above polypeptides.
In a further aspect, the present invention provides a substance which is a fragment of a polypeptide having the amino acid sequence set out in
FIG. 1A
or
1
F which exhibits a biological prop
Bonny Christophe
Waeber Gerard
Dann Dorfman Herrell and Skillman
Nolan Patrick J.
Waeber Gerard
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