Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-07-31
2001-05-15
Allen, Marianne P. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C536S024300, C536S024310, C536S024330, C435S006120, C435S069100, C435S091200, C435S320100, C435S440000, C435S455000, C435S471000, C435S476000
Reexamination Certificate
active
06232459
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a new synaptojanin isoform and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, and neurological and immune disorders.
BACKGROUND OF THE INVENTION
Vesicle transport is the general process in eukaryotic cells by which proteins synthesized in the endoplasmic reticulum (ER) are transported via the Golgi network to the various compartments in the cell where they will function. Other proteins are transported to the cell surface by this process where they may be secreted (exocytosis). Such proteins include membrane bound receptors or other membrane proteins, neurotransmitters, hormones, and digestive enzymes. The transport process uses a series of transport vesicles that shuttle a protein from one membrane-bound compartment (donor compartment) to another (acceptor compartment) until the protein reaches its proper destination (Rothman, J. E and Wieland, F. T. et al. (1996) 727:227-33). Endocytosis is the reverse process by which cells internalize nutrients, solutes or small particles (pinocytosis) or large particles such as internalized receptors, viruses, bacteria, or bacterial toxins (phagocytosis).
Transport vesicles of various types are formed from specialized coated regions of membranes that bud off as coated vesicles with a distinctive cage of proteins surrounding the vesicle. The nature of the protein coat defines the transport vesicle in terms of the types of molecules that are transported and their destination. Clathrin-coated vesicles, for example, selectively transport transmembrane receptors between the ER and the plasma membrane while coatomer-coated vesicles mediate non-selective transport of various molecules from the ER and the Golgi network. Synaptic vesicles are a highly specialized type of transport vesicle that neurons use to secrete neurotransmitters at the neural synapse. Following secretion, the synaptic vesicle membranes are internalized and reused for further neurotransmitter release. The process of synaptic vesicle recycling involves the interaction of various proteins, three of which are synaptojanin, dymanin, and amphiphysin (Ramjaun, A. R. and McPherson, P. S. (1999) J. Biol. Chem. 271:24856-61). Synaptojanin and dynamin were first identified as major Src homology 3 (SH3) domain-binding proteins in brain. In particular, synaptojanin and dynamin both interact with the SH3 domains of amphiphysin, a nerve terminal protein that is implicated in synaptic vesicle endocytosis. These SH3 interactions may play a role in subcellular targeting of synaptojanin and dynamin to specific sites of synaptic vesicle endocytosis on the plasma membrane (Ramjaun et al. supra).
Synaptojanin (Syn) is a 145 kDa protein that contains (1) a region in the N terminus that is homologous with various inositol phosphatases, and (2) a proline-rich C terminus containing numerous consensus sites for SH3 binding (McPherson, P. S. et al. (1996) Nature 379:353-57). Inositol polyphosphates are believed to play a role in endocytosis and in other aspects of membrane trafficking. A proline-rich consensus sequence for SH3 binding is represented as Xp&phgr;PpXP; in which X is any amino acid residue, P is a conserved proline residue, and p and &phgr; (lower case) indicate a preference for proline and hydrophobic residues, respectively (Hongtao, Y. et al. (1994) Cell 76:933-45). In addition to the 145 kDa isoform of Syn, a 170 kDa isoform of the protein has been identified (Ramjaun et al. supra; McPherson et al. supra). The 170 kDa isoform results from the addition of a 28 kDa polypeptide to the C terminus of the 145 kDa isoform. This added 266 amino acid sequence is rich in proline residues and contains additional SH3 domain-binding consensus sequences. The 28 kDa polypeptide is encoded by a second open reading frame (ORF) normally separated from a first ORF by a stop codon. Expression of the larger 170 kDa isoform is believed to result from alternative splicing of the cDNA which deletes the stop codon (McPherson et al. supra).
The presence of the 28 kDa sequence alters the properties of Syn in two important ways: 1) while the 145 kDa isoform is expressed almost exclusively in adult rat brain, the 170 kDa isoform is absent from adult brain and widely expressed in non-neuronal tissues; and 2) the 170 kDa isoform is more strongly membrane bound than the 145 kDa isoform. These properties may allow the 170 kDa isoforn of Syn to play a unique and perhaps more general role in endocytosis (McPherson et al. supra).
The discovery of a new synaptojanin isoform and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, and neurological and immune disorders.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, synaptojanin isoform (NSYN-1), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding NSYN-1 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified NSYN-1 having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neurological disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified NSYN-1.
The invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of NSYN-1.
The invention also provides a method for treating or preventing an immune disorder comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of NSYN-1.
The invention also provides a method for detecting a polynucleotide which encodes NSYN-1 in a biological sample comprising the steps of: a) hybridizing the complement of the polynuc
Lal Preeti
Tang Y. Tom
Allen Marianne P.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Zeman Mary K
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