25312, a novel human agmatinase-like homolog

Details 25312, a novel human agmatinase-like homolog 25312, a novel human agmatinase-like homolog

2001-02-22

2003-11-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S226000, C536S023200

Reexamination Certificate

active

06642039

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a newly identified human agmatinase-like arginase, designated “25312”. The invention also relates to polynucleotides encoding the agmatinase-like arginase. The invention further relates to methods using the agmatinase-like polypeptides and polynucleotides as a target for diagnosis and treatment in disorders mediated by or related to the agmatinase-like arginase. The invention further relates to drug-screening methods using the polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the polypeptides and polynucleotides. The invention further relates to agonists and antagonists identified by drug screening methods with the polypeptides and polynucleotides as a target.
BACKGROUND OF THE INVENTION
Human agmatinase is an enzyme that plays a role in the hydrolysis of agmatine [4-(aminobutyl)guanidine] to putrescine and other polyamines such as spermine and spermidine which are essential for DNA replication, cell homeostasis and cell transformation. Polyamines are required for entry and progression of the cell cycle. Also, augmentation of polyamine levels is essential for cellular transformation. Agmatine is a metabolite of arginine via arginine decarboxylase (ADC) and is implicated in the attenuation of cellular polyamine levels (Satriano et al. (1998)
J. Biol. Chem
. 273 (25): 15313-15316). Agmatine represents an alternate pathway to polyamine production in contradistinction to the well-studied pathway mediated by ornithine decarboxylase (ODC) which acts in the metabolism of arginine to yield putrescine which feeds into polyamine synthesis. End-products of arginine metabolism include the cell-signaling molecules: NO, glutamate, and agmatine. (Wu et al., (1998)
Biochem. J
. 336: 1-17). Mammalian ADC is membrane associated and expressed in the inner membranes of mitochondria.
Agmatine is widely and unevenly distributed in a variety of mammalian tissues including serum. The tissues where agmatine has been identified include: stomach, aorta, small intestine, large intestine, spleen, lung, vas deferens, adrenal gland, kidney, heart, liver, skeletal muscle, testes, and brain. The highest concentration was found in stomach, aorta, and small intestine (Raasch et al., (1995)
Life Sciences
56 pp. 2319-2330). Agmatine binds to &agr;
2
-adrenergic and imidazoline receptors and is bioactive in a number of tissues (Wu et al., (1998)
Biochem. J
336:1-17). It is contained in neurons and is found in serum which is consistent with its role as a putative neurotransmitter and/or a hormone. Agmatine potentiates the analgesic effects of morphine and clonidine in a dose-dependent manner and decreases the EC
50
of morphine and clonidine by more than 75% in a mouse tail-flick test. Intrathecal agmatine at high doses causes a decrease in the pain threshold (Jin, Li et al., (1999)
Acta Pharmacologica Sinica
20 (1): 81-85).
The enzyme has been isolated from rat brain and was localized primarily in the mitochondria wherein it degrades the substrate, agmatine at its site of action (Regunathan et al.,(1996)
J. Neurochem
. 67(4): 1761-65). Specifically, the enzyme is localized in the mitochondrial matrix.
It is presumed that agmatine is a biologically active molecule with numerous physiological roles including, but not limited to: binding to &agr;
2
-adrenergic and imidazoline receptors, causing release of catecholamine from adrenal chromaffin cells, stimulating release of insulin and uptake of Ca in pancreatic cells, inhibitor of lipolysis in rat adipocytes, increase glucose uptake and glycogen content of the rat diaphragm, and increase glucose oxidation and lipogenesis in fat pads and glucose oxidation in isolated fat cells (Raasch et al., (1995)
Life Sciences
56 pp. 2319-2330).
The concentration of agmatine in the whole brain is comparable to that of other neurotransmitters. It is unevenly distributed with the highest concentration in the hypothalamus, forebrain, and cerebral cortex (Reis et al. (1999)
Annals of the NY Academy of Sciences
881: 65-80). Agmatine is synthesized and stored in astrocytes (Youngson et al.,
Ann. NY Acad. Sci.
763: 440-444).
Agmatinase (agmatine ureohydrolase) is an enzyme that hydrolyzes agmatine to form putrescine and urea. Putrescine along with spermine and spermidine are polyamines.
Polyamines such as putrescine, spermidine, and spermine are required for DNA replication, proliferation, and cell homeostasis. Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of polyamine biosynthesis and one of the most highly regulated eukaryotic enzymes. Cellular polyamine transporters are stimulated by many of the same factors that induce ODC activity. Cellular polyamine uptake occurs both in normal and rapidly proliferating cells and tumor lines (Moulinoux et al. (1991)
Cell. Mol. Biol
. 37: 773-783; Bogle et al. (1994)
Am. J Physiol
. 266: C776-C783; Holley et al. (1992)
Cancer Res
. 52:4190-4195).
Polyamines have been reported in the herpes simplex virion (HSV) (Gibson et al. (1973)
Polyamines in Normal and Neoplastic Growth
, edited by D. H. Russell, Raven Press, NY). The polyamines may serve as specific structural components of the virion and serve to neutralize the electronegativity of DNA. Agmatinase may play a role in HSV infection as it is induced during the latent phase of HSV replication (unpublished data). Also, 25312 expression is induced during infection by the DNA virus HBV (unpublished data). Thus, high levels of polyamine synthesis via the agmatinase pathway may be a requirement for DNA viruses.
Intracellular polyamine concentrations are autoregulated by the induction of the protein antizyme (Matsufuji et al. (1995)
Cell
80:51-60). Antizyme binds to ODC and inhibits its activity and accelerates its degradation (Hayashi et al. (1996)
Trends Biochem Sci
. 21:27-30). More recently, antizyme has been shown to suppress polyamine transporters (Mitchell et al. (1994)
Biochem. J
. 299:19-22; Suzuki (1994)
Proc. Natl. Acad. Sci
. 91:8930-8934). Thus, antizyme through its ability to suppress both the polyamine biosynthetic enzyme ODC and polyamine transporters is an effective endogenous mechanism for limiting intracellular polyamine levels.
Recent research has demonstrated the induction of antizyme by agmatine. The induced antizyme can bind to ornithine decarboxylase (ODC) and depress polyamine biosynthesis and transport. (Satriano et al. (1998)
J. Biol. Chem
. 273: 15313-15316). The capacity of agmatine to induce antizyme is demonstrated by (a) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (b) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor (Satriano et al. (1998)
J. Biol. Chem
. 273 (25):15313-15316).
There is evidence that agmatine has several potential roles in mammalian physiology, including: acting as a neurotransmitter, as a secretogogue and as an endogenous inhibitor of all isoforms of NOS, and it may play a role in modulating the state of macrophage activation during inflammation by regulating NOS activity and NO production (Sastre et al. (1998)
Biochem. J
. 330:1405-1409).
Agmatine (AGM) has long been characterized as a constituent of bacteria, plants and some invertebrates (Tabor and Tabor (1984)
Ann. Rev. Biochem
. 53:749-790). More recently, agmatine was shown to be expressed in rat brain (Li, G. et al. (1994)
Science
263:966-969). Agamatine is an endogenous ligand at imidazoline and &agr;-adrenergic receptors to which it binds with high affinity (Tabor et al. (1984)
Ann. Rev. Biochem
. 53:749-790). Agmatine also has properties of an endogenous neurotransmitter. However, its actual role in normal brain function has not yet been established (Reis et al. (1998)
Adv Pharmacol
42:645-9). Agmatine is locally synthesized in the brain and stored in a large number of neurons with selective distribution in the central nervous system. Also, it can be enzym

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