27411, a novel human PGP synthase

Details 27411, a novel human PGP synthase 27411, a novel human PGP synthase

2001-02-28

2002-10-15

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S252300, C435S320100, C435S071100, C435S325000, C536S023200

Reexamination Certificate

active

06465230

ABSTRACT:

FIELD OF THE NVENTION
The present invention relates to newly identified human phosphatidyl-glycerolphosphate (PGP) synthase belonging to the family of mammalian PGP synthases. The invention also relates to polynucleotides encoding the PGP synthase. The invention further relates to methods using the PGP synthase polypeptides and polynucleotides as a target for diagnosis and treatment in PGP synthase-mediated or -related disorders. The invention further relates to drug-screening methods using the PGP synthase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the PGP synthase polypeptides and polynucleotides. The invention further relates to procedures for producing the PGP synthase polypeptides and polynucleotides.
BACKGROUND OF THE NVENTION
Cardiolipin is a dimeric phospholipid which plays an important role in mitochondrial biogenesis and function. It is required for activity of several mitochondrial enzymes and possibly for the transport of proteins into the mitochondria in eukaryotes (Minskoff, S. et al. (1997)
Biochimica et Biophysica Acta
1348: 187-191). Cardiolipin appears to be involved either directly or indirectly, in the modulation of a number of cellular processes including the activation of mitochondrial enzymes and the production of energy by oxidative phosphorylation (Hatch, G. (1998)
International J. of Mol. Medicine
1: 33-41).
Cardiolipin is found in animals, plants, and fungi. In mammals it is found exclusively in mitochondria. Cardiolipin is the principal polyglycerophospholipid found in the heart and most mammalian tissues (Hatch, G. (1998)
International J. of Molec. Medicine
1: 33-41). The biosynthetic pathway of cardiolipin has been well studied in yeasts. The first enzyme in the cardiolipin biosynthetic pathway is phosphatidylglycerolphosphate synthase (PGP synthase). PGP synthase is a key enzyme in the pathway as it catalyzes the committed first step in the pathway.
The biosynthesis of cardiolipin occurs in 3 enzymatic steps. In the first step, PGP synthase catalyzes the formation of phosphatidylglycerolphosphate (PGP) from phosphatidyl-CMP (CDP-diacylglycerol, CDP-DG) and glycerol 3-phosphate. PGP is then dephosphorylated to phosphatidylglycerol (PG) by PGP phosphatase. Finally, in eukaryotes cardiolipin is synthesized from PG and another molecule of CDP-DG in a reaction catalyzed by cardiolipin synthase.
Cardiolipin appears to be essential for the function of several enzymes of oxidative phosphorylation. (Hatch, G. (1996)
Molecular and CellularBiochemistry
159:139-148). Also, cardiolipin has been implicated in the role of many enzymatic activities, including but not limited to: (1) cytochrome c oxidase, (2) camitine acylcarnitine translocase, (3) mitochondrial protein import, and (4) binding of matrix Ca
+2
(Kawasaki, K. (1999)
J. of Biological Chemistry,
Vol. 274, No.3, 1828-1834).
There must be stringent levels of control of the enzymes involved in cardiolipin metabolism in the heart in order to maintain the appropriate content and molecular species composition of the phospholipid. The maintenance of cardiolipin content and molecular composition in cardiac mitochondria is essential for proper cardiac function (Hatch, G. (1998)
International J. of Mol. Medicine
1:33-41).
Phosphatidylglycerol (PG) and cardiolipin (CL) are the most widely distributed glycerophosphatides in the membrane lipids of animals, plants and microbes (Hostletler, K. Y. (1982) in
Phospholipids
(Hawthorne and Ansell, eds) pp.215-261, Elsevier/North Holland Biomedical Press, Amsterdam).
PG is localized in many intracellular locations as a component of phospholipids, representing less than 1% of total lipid phosphorous, except in the lung where it represents about 10% of the total phospholipids (Mason, R. J. et al., (1980)
Biochim. Biophys. Acta
617: 36-50). PG serves as an important component of the pulmonary surfactant in the lung (Ohtsuka et al., (1993)
J. Biol. Chem.
Vol. 268:22908-22913). CL is localized primarily in the mitochondria and appears to be essential for the function of several enzymes of oxidative phosphorylation. CL is essential for production of energy for the heart to beat (Hatch, G. M. (1996)
Molecular and Cellular Biochemistry,
159: 139-148).
PGP synthase has been extensively studied and characterized in two evolutionarily divergent yeasts,
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe.
PGP synthase has been purified to homogeneity from
S. pombe
(Minskoff, S. et al. (1997)
Biochimica et Biophysica Acta
1348: 187-191). In contrast to the second and third enzymes of the cardiolipin biosynthetic pathway, PGP synthase activity is highly regulated both by cross-pathway control and by factors affecting mitochondrial development.
PGP synthase has been shown to be controlled by two sets of factors: cross-pathway control and factors affecting mitochondrial development. Cross-pathway control of phosphatidylinositol and phosphatidylcholine control is characterized by three parameters. First, the availability of the water-soluble phospholipid precursor inositol controls expression of phospholipid biosynthetic enzymes. Second, inositol repression of phospholipid biosynthesis occurs only if cells can synthesize phosphatidyl-choline. Third, inositol repression is mediated by the INO2-INO4-OPI1 regulatory genes. PGP synthase is regulated by inositol. However, it is not subject to control by the INO2-INO4-OPI1 regulatory genes. PGP synthase activity is decreased 3-5 fold in
Saccharomyces cerevisiae
cells grown in the presence of inositol (Greenberg, M. L. et al., (1988)
Mol. Cell. Biol.
8: 4773-4779).
PGP synthase is commonly referred to as glycerophosphate phosphatidyl-transferase (E.C. 2.7.8.5). It catalyzes a substituted phospho group transfer. The natural substrate of the enzyme is CDP-1,2-diacyl-sn-glycerol and glycerol 3-phosphate (involved in the synthesis of phosphatidylgylcerol). Different cofactors and prosthetic groups which have been shown to be important for maximal PGP synthase activity include, but are not limited to: Triton X-100, phosphatidylethanolamine and phosphatidylinositol. Different metal/salts which have been shown to be important for PGP synthase activity include, but are not limited to: Mn
+2
, Mg
+2
, Ca
+2
, Co
+2
, and Ba
+2
.
PGP synthases in two different yeasts (
S. cerevisiae
and
S. pombe
) were found to be sensitive to thioreactive compounds and have a requirement for divalent cations (Minskoff, S. et al. (1997)
Biochimica et Biophysica Acta
1348: 187-191).
Inhibitors of PGP synthase have been shown to include, but are not limited to: liponucleotide, CDPdiacylglycerol, glycerol 3-phosphate, thioreactive agents, calcium, inositol, Triton X-100, magnesium, cadmium, zinc, copper, and mercury (see www.expasy.ch/cgi-bin/enzyme-search-ec). As one example, PGP synthase activity was shown to decrease 3 to 5 fold in
S. cerevisiae
cells grown in the presence of inositol.
PGP synthase activity can be assayed by determining the conversion of [
14
C(U)] glycerol 3-phosphate to phosphatidyl [
14
C(U)]glycerol 3-phosphate as described by Cao et al. (Cao et al. (1994)
LIPIDS,
Vol. 29, no.7, pp.475-480).
Chinese hamster ovary (CHO) cells defective in PGP synthase production have been studied to better elucidate the role of the enzyme in the biosynthesis of PG and CL (Ohtsuka, T. et al., (1993)
J. Biol. Chem. Vol.
268, No. 30, pp. 22908-22913). Ohtsuka et al. developed a rapid autoradiographic screening assay for detecting PGP synthase activity in the lysates of Chinese hamster ovary cell colonies immobilized on polyester, as described by Raetz et al. (Raetz et al., (1982)
Proc. Natl. Acad. Sci. U.S.A.
79: 3223-3227). The Ohtsuka study confirmed the role of PGP synthase in the biosynthesis of PG and its essential role in the growth of CHO cells. The results provided direct evidence for the formation of PG in vivo and that PG is a major metabolic p

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