Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1999-09-16
2001-09-04
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C530S350000
Reexamination Certificate
active
06284505
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to amino transporters from mammalian species and the genes corresponding to such transporters. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel human amino acid transporter gene. The invention also relates to the construction of recombinant expression constructs comprising such cDNAs from a novel human amino acid transporter gene of the invention, said recombinant expression constructs being capable of expressing amino acid transporter protein in cultures of transformed prokaryotic and eukaryotic cells as well as in amphibian oocytes. Production of the transporter protein of the invention in such cultures and oocytes is also provided. The invention relates to the use of cultures of such transformed cells to produce homogeneous compositions of the novel transporter protein. The invention also provides cultures of such cells and oocytes expressing transporter protein for the characterization of novel and useful drugs. Antibodies against and epitopes of the transporter protein are also provided by the invention.
2. Background of the Invention
The approximately 20 naturally-occurring amino acids are the basic building blocks for protein biosynthesis. Certain amino acids, such as glutamate and glycine, as well as amino acid derivatives such as &ggr;-aminobutyric acid (GABA), epinephrine and norepinephrine, and histamine, are also used as signaling molecules in higher organism such as man. For these reasons, specialized trans-membrane transport proteins have evolved in all organisms to recover or scavenge extracellular amino acids (see Christensen, 1990
, Physiol. Rev
. 70: 43-77for review).
These transporter proteins play a particularly important role in uptake of extracellular amino acids in the vertebrate brain and peripheral motor and sensory tissues (see Nicholls & Attwell, 1990, TiPS 11: 462-468). Amino acids that function as neurotrarsmitters must be scavenged form the synaptic cleft between neurons to enable continuous repetitive synaptic transmission. More importantly, it has been found that high extracellular concentrations of certain amino acids (including glutamate and cysteine) can cause neuronal cell death. High extracellular amino acid concentrations are associated with a number of pathological conditions, including ischemia, anoxia and hypoglycemia, as well as chronic illnesses such as Huntington's disease, Parkinson's disease, Alzheimer's disease, epilepsy and amyotrophic lateral sclerosis (ALS: see Pines et al., 1992
Nature
360: 464-467).
Glutamate is one example of such amino acid. Glutamate is an excitatory neurotransmitter (i e., excitatory neurons use glutamate as a neurotransmitter). When present in excess (>about 300 &mgr;M; Bouvier et al., 1992
, Nature
360: 471-474; Nicholls & Attwell, ibid.; >5 &mgr;M for 5 min.; Choi et al., 1987
, J. Neurosci
. 7: 357-358), extracellular glutamate causes neuronal cell death. Glutamate transporters play a pivotal role in maintaining non-toxic extracellular concentrations of glutamate in the brain. During anoxic conditions (such as occur during ischemia), the amount of extracellular glutamate in the brain rises dramatically. This is in part due to the fact that, under anoxic conditions, glutamate transporters work in reverse, thereby increasing rather that decreasing the amount of extracellular glutamate found in the brain. The resulting high extracellular concentration of glutamate causes neuron death, with extremely deleterious consequences for motor and other brain functions, resulting in stroke and other instances of organic brain dysfunction.
This important role for amino acid transporters in maintaining brain homeostasis of extracellular amino acid concentrations has provided the impetus for the search for an development of compounds to modulate and control transporter function. However, conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-transporter) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. These same limitations arise in the use of animal-derived sensory tissue, particularly retina, to study the effects of transporter function in these tissues. The ability to synthesize human transporter molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds.
Amino acid transporters are known in the art, and some of these proteins have been isolated biochemically and their corresponding genes have been recently cloned using genetic engineering means.
Christensen et al., 1967
, J. Biol. Chem
. 242: 5237-5246 report the discovery of a neutral amino acid transporter (termed the ACS transporter) in Erlich ascites tumor cells.
Makowske & Christensen, 1982
, J. Biol Chem
. 257: 14635-14638 provide a biochemical characterization of hepatic amino acid transport.
Kanner & Schuldiner, 1987, CRC
Crit. Rev. Biochem
. 22: 1-38 provide a review of the biochemistry of neurotransmitters.
Olney et al., 1990
Science
248: 596-599 disclose that the amino acid cysteine is a neurotoxin when present in excess extracellularly.
Wallace et al., 1990
, J. Bacteriol
. 112: 3214-3220 report the cloning and sequencing of a glutamate/aspartate transporter gene termed gltP from
Escherichia coli
strain K12.
Kim et al., 1991
, Nature
352: 725-728 report the discovery that a cationic amino acid transporter is the cell surface target for infection by ecotropic retroviruses in mice.
Wang et al., 1991
, Nature
352: 729-731 report the discovery that a cationic amino acid transporter is the cell surface target for infection by ecotropic retroviruses in mice.
Maenz et al., 1992
, J. Biol Chem
. 267: 8330-8335 report that the ASC transporter acts in a electrochemically neutral manner so that sodium ion co-transport occurs without disrupting the normal membrane potential of the cells expressing the transporter.
Engelke et al., 1992
, J. Bacteriol
. 171: 5551-5560 report cloning of a dicarboxylate carrier from
Rhizobium meliloti.
Guastella et al., 1992
, Proc. Natl. Acad. Sci. USA
89: 7189-7193 disclose the cloning of a sodium ion and chloride ion-dependent glycine transporter from a glioma cell line that is expressed in the rat forebrain and cerebellum.
Kavanaugh et al., 1992
, J. Biol Chem
. 267: 22007-22009 report that biochemical characterization of a rat brain GABA transporter expressed in vitro in
Xenopus laevis
oocytes.
Storck et al., 1992
, Proc. Natl. Acad. Sci. USA
89: 10955-10959 disclose the cloning and sequencing of a sodium ion-dependent glutamate/aspartate transporter from rat brain termed GLAST1.
Bouvier et al., ibid., disclose the biochemical characterization of a glial cell-derived glutamate transporter.
Pines et al., ibid., report the cloning and sequencing of a glial cell glutamate transporter from rat brain termed GLT-1.
Kanai & Hediger, 1992
, Nature
360: 467471 disclose the cloning and sequence of a sodium ion-dependent neutral amino acid transporter of the A type that is homologous to a sodium-ion dependent glucose transporter.
Arriza et al., 1994
, J. Neurosci
. 14: 5559-5569 disclose genes for three novel glutamate transporters.
Nicholls & Attwell, ibid., review the role of amino acids and amino acid transporters in normal and pathological brain functions.
In humans, the sodium-dependent glutamate uptake transporters include 4 known subtypes, termed EAAT1 through EAAT3, that are expressed in neurons in the brain, as disclosed in co-owned and co-pending U.S
Amara Susan G.
Arriza Jeffrey L.
Eliasof Scott
Kavanaugh Michael P.
Achutamurthy Ponnathapu
Kerr Kathleen
McDonnell & Boehnen Hulbert & Berghoff
Oregon Health & Science University
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