Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-08-04
2001-02-13
Nolan, Patrick (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023500
Reexamination Certificate
active
06187563
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to proteins, polypeptides, or fragments thereof, which interact with autoantigens of autoimmune diseases, such as type I diabetes. More particularly the invention relates to polypeptides, or fragments thereof, which interact with human ICA512 and phogrin. The invention also relates to nucleic acid sequences which encode these polypeptide or polypeptide fragments. The invention also relates to methods for identifying and treating individuals who suffer from or are susceptible to diabetes, screening for autoimmune diseases such as type I diabetes, and modulating hormone and neuropeptide secretion using proteins or protein fragments which interact with autoantigens of autoimmune diseases.
2. Description of the Related Art
Insulin dependent diabetes mellitus (also known as IDDM or type I diabetes) primarily afflicts young people. Although insulin is available for treatment, the several-fold increased morbidity and mortality associated with this disease urge the development of early diagnostic and preventive methods. The destruction of pancreatic &bgr;-cells, which precedes the clinical onset of IDDM, is mediated by autoimmune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high increase of circulating &bgr;-cell-specific autoantibodies at the time of diagnosis.
A major goal of diabetes research has been to develop immune interventions that block or inhibit the destruction of &bgr;-cells and development of IDDM. For example, U.S. Pat. No. 5,512,447, the disclosure of which is incorporated herein by reference, describes assays for the detection of diabetes and prediabetic status by exposing patient serum samples to purified ligand capable of binding autoantibodies specific for a 64 kD autoantigen present in pancreatic &bgr;-cells. One of the most intriguing observations resulting from studying the cell biology of &bgr;-cell autoantigens is that all major targets of IDDM autoantibodies identified thus far are directly connected with the secretory apparatus of &bgr;-cells (Solimena, Diabetes, Metab. Rev. 14:227-240 (1994)).
A large body of evidence indicates that protein and lipid phosphorylation participates in the trafficking of secretory granules (SGs), synaptic vesicles (SVs), and synaptic-like microvesicles (SLMVs) of neuroendocrine cells, including pancreatic &bgr;-cells. Several membrane and cytosolic phosphoproteins involved in priming, docking and fusion of regulated secretory vesicles (Ferro-Novick, S. et al., Nature 370:191-193 (1994); Martin, T. F., Curr. Opin. Neurobiol. 4:626-632 (1994); Calakos, N. et al., Physiol. Rev. 76:1-29 (1996)) have been shown to be substrates for serine/threonine phosphorylation (Greengard, P. et al., Science 259:780-785 (1993); Ashcroft, F. M. et al., J. Cell Biochem. 55 Supp:54-65 (1994)). However, less evidence is available implicating tyrosine phosphorylation in these processes. The following evidence suggests that tyrosine phosphorylation may play a role in regulated secretion.
The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A both stimulate insulin secretion in neonatal islets (Sorenson, R. L. et al., Endocrinol. 134:1975-1978 (1994)), and genistein has been shown to affect the ATP-dependent priming of SGs in semi-intact PC12 cells (Hay, J. C. et al., J. Cell Biol. 119:139-151 (1992)). In GH3 cells, both PTK and protein tyrosine phosphatase (PTP) inhibitors have been shown to impair the biogenesis of SGs from the trans-Golgi network (Austin, C. D. et al., J. Cell Biol. 135:1471-1483 (1996)) as well as modulate the activity of L-type Ca
2+
channels (Cataldi, M. et al., J. Biol. Chem. 271:9441-9446 (1996)) which are known to be coupled to neurosecretion.
Synaptophysin and synaptogyrin, two intrinsic membrane proteins of SVs, have both been shown to be tyrosine phosphorylated (Pang, D. T. et al., Proc. Natl. Acad. Sci. U.S.A. 85:762-766 (1988); Stenius, K., et al., J. Cell Biol. 131:1801-1809 (1995)), but the physiological relevance has not yet been determined. Furthermore, PTK pp60
c-src
has been shown to be peripherally associated with regulated secretory vesicles, including SGs of chromaffin cells (Parsons, S. J. et al., Biochem. Biophys. Res. Comm. 134:736-742 (1986); Grandori, C. et al., J. Cell Biol. 107:2125-2135 (1988)).
Annexins (soluble Ca
2+
and phospholipid-binding proteins) have been implicated in exocytosis in different cell types, including endocrine cells (Burgoyne, R. D. et al., J. Anat. 183:309-314 (1993)). Annexin II, in particular, has been shown to bind chromaffin SGs and to reconstitute secretion from permeabilized chromaffin cells of the adrenal medulla (Sarafian, T. et al., C.J. Cell Biol. 114:1135-1147 (1991)), and has been shown to be tyrosine phosphorylated by 60
c-src
(Hubaishy, I. et al., Biochemistry 34:14527-14534 (1995)). pp60
c-src
has also been shown to phosphorylate botulinum A and E (Ferrer-Montiel, A. V. et al., J. Biol. Chem. 271:18322-18325 (1996)), two clostridium neurotoxins which cause the block of neurosecretion and paralysis by cleaving the synaptobrevin binding protein SNAP-25 at neuromuscular junctions (Tonello, F. et al., Adv. Exp. Med. Biol. 389:251-260 (1996)). Since tyrosine phosphorylation enhances the protease activity of both botulinum A and E, it is possible that tyrosine phosphorylation and regulated secretion are coupled in certain cells.
Islet cell autoantigen 512 (ICA512, also known as IA-2 and PTP35), is an intrinsic membrane protein of SGs which is expressed in virtually all neuroendocrine cells including peptide-secreting endocrine cells, neurons of the autonomic nervous system, and neurons of the hypothalamus and the amygdala in the brain (Solimena, M. et al., EMBO J. 15:2102-2114 (1996)). Human ICA512 has been identified as an autoantigen of IDDM (Rabin, D. U. et al., Diabetes 41:183-186 (1992); Rabin, D. U. et al., J. Immunol. 152:3183-3188 (1994); Lan, M. S. et al., DNA Cell Biol. 13:505-514 (1994)), and has been postulated to play a role in regulated peptidergic secretion from neuroendocrine cells, including insulin-secreting cells of the pancreatic islets. The open reading frame of human ICA512 encodes a protein of 979 amino acids (FIG.
1
). The sequence includes a signal peptide (residues 1-34) and two putative N-glycosylation sites (residues 506 and 524) in the extracellular domain (residues 1-575, henceforth defined as ectodomain), a single transmembrane domain (residues 576-600), and a cytoplasmic domain (residues 601-979) which includes a region displaying homology to protein tyrosine phosphatases (PTPs) (residues 696-979). Like several other enzymatically active receptor protein tyrosine phosphatases (RPTPs) (Streuli, M. et al., EMBO J. 11:897-907 (1992); Serra-Pages, C. et al., J. Biol. Chem. 269:23632-23641 (1994); Brady-Kalnay, S. M. et al., J. Biol. Chem. 269:28472-28477 (1994); Pulido, R. et al, Proc. Natl. Acad. Sci. U.S.A. 92:11686-11690 (1995)), ICA512 is processed within its ectodomain (Solimena, M. et al., EMBO J. 15:2102-2014 (1996); Hermel et al., Eur. J. Neurosci. 11:20690 (1999)). Cleavage of ICA512 (arrow in
FIG. 1
) generates a 65 kD transmembrane fragment (residues 449-979) which remains associated with SG membranes, and a putative N-terminal fragment (residues 35-448).
The cytoplasmic domain of ICA512 is homologous with PTPs and suggests that ICA512 participates in signal transduction pathways involving tyrosine phosphorylation. Several features, however, distinguish ICA512 from conventional RPTPs.
First, the ectodomain of human ICA512 does not contain any of the motifs found in most RPTPs, including Ig domains, type III fibronectin repeats, MAM (meprin, A5, PTP&mgr;) domains, or carbonic anhydrase-like motifs, all of which are thought to mediate cell—cell or cell-matrix contact (Brady-Kalnay, S. M. et al., Curr. Opin. Cell Biol. 7:650-657 (1995); Streuli, M. Curr. Opin. Cell Bio. 8:182-188 (1996)). (The RGD motif of the type III fibronectin repeat, however, is present once in the
Berghs Constance A. F. M.
Dirkx, Jr. Ronald A.
Solimena Michele
Ewoldt Gerald R.
Garabedian Todd E.
Nolan Patrick
Wiggin & Dana
Yale University
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