Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-03-27
2002-04-30
Saidha, Tekchand (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S198000, C435S252300, C435S320100, C536S023200, C536S023500, C536S023600, C530S350000
Reexamination Certificate
active
06379665
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of molecular biology and particularly relates to nucleic acid sequences that encode novel phospholipases.
BACKGROUND
The mechanism by which specificity of physiological responses are conferred by a limited number of signal transducing substances, typically enzymes, is poorly understood. Cellular receptors on the surfaces of various cells are involved and initiate multiple signaling pathways. Some of the receptors on neutrophils are known: the PAF receptor, the interleukin-8 receptor and the fMetLeuPhe receptor all belong to the super-family of G-protein-linked receptors. A common feature of these receptors is that they span the cell membrane seven times, forming three extracellular and three intracellular loops and a cytoplasmic carboxy-terminal tail. The third loop and the tail exhibit extensive variability in length and sequence, leading to speculation that these parts are responsible for the selective interaction with the various G-proteins. Many of these G-protein-linked receptors stimulate the activation of three phospholipases, phospholipase C (PLC), phospholipase D (PLD) and phospholipase A
2
(PLA
2
). These phospholipases constitute a family of regulatory enzymes which trigger various neutrophilic functions, for example adherence, aggregation, chemotaxis, exocytosis of secretory granules and activation of NADPH oxidase, i.e., the respiratory burst.
The main substrates for the phospholipases are membrane phospholipids. The primary substrates for PLC are the inositol containing lipids specifically and typically phosphotidylinositol (PI). PI is phosphorylated by PLC resulting in the formation of PIP, phosphotidylinositol 4-phosphate. The primary substrate for PLD and PLA
2
is phosphatidylcholine (PC), a relatively ubiquitous constituent of cell membranes. The activity of cytosolic PLA
2
on PC liberates arachidonic acid, a precursor for the biosynthesis of prostaglandins and leukotrienes and possible intracellular secondary messenger. PLD, on the other hand, catalyzes the hydrolytic cleavage of the terminal phosphate diester bond of glycerophospholipids at the P-O position. PLD activity was originally discovered in plants and only relatively recently discovered in mammalian tissues. PLD has been the focus of recent attention due to the discovery of its activation by fMetLeuPhe in neutrophils. PLD activity has been detected in membranes and in cytosol. Although a 30 kD (kilodalton) and an 80 kD activity have been detected, it has been suggested that these molecular masses represented a single enzyme with varying extents of aggregation. See Cockcroft,
Biochimica et Biophysica Acta
1113: 135-160 (1992). One PLD has been isolated, cloned and partially characterized. See Hammond,
J. Biol. Chem.
270:29640-43 (1995). Biological characterization of PLD1 revealed that it could be activated by a variety of G-protein regulators, specifically PKC (protein kinase C), ADP-ribosylation factor (ARF), RhoA, Rac1 and cdc-42, either individually or together in a synergistic manner, suggesting that a single PLD participates in regulated secretion in coordination with ARF and in propagating signal transduction responses through interaction with PKC, PhoA and Rac1. Nonetheless, PKC-independent PLD activation has been associated with Src and Ras oncogenic transformation, leaving open the possibility that additional PLDs might exist. See Jiang,
Mol. and Cell. Biol.
14:3676 (1994) and Morris,
Trends in Pharmacological Sciences
17: 182-85(1996). The difficulty may arise at least in part from the fact that in the phospholipase family enzymes may or may not be activated by, and catalyze, multiple substances, making sorting, tracking and identification by functional activities impractical.
There exists a need in the art for the identification and isolation of phospholipase enzymes. Without such identification and isolation, there is no practical way to develop assays for testing modulation of enzymatic activity. The availability of such assays provides a powerful tool for the discovery of modulators of phospholipase activity. Such modulators would be excellent candidates for therapeutics for the treatment of diseases and conditions involving pathological mitogenic activity or inflammation.
SUMMARY OF THE INVENTION
In one aspect, the invention provides novel mammalian phospholipase D (PLD) proteins, which are substantially free from other proteins with which they are typically found in their native state. These novel mammalian PLD proteins include polypeptides substantially free of association with other polypeptides and comprising an enzyme of mammalian origin having a phosphatidylcholine-specific pohspholipase D activity and containing at least two copies of the amino acid motif HXKXXXXD. More specifically, these proteins include polypeptides substantially free of association with other polypeptides and comprising PLD polypeptides that are perinuclear membrane associated, require PI(4,5)P2 for in vitro activity and are activated by one or more G-proteins. Alternatively, these proteins include polypeptides substantially free of association with other polypeptides and comprising PLD polypeptides that are plasma membrane associated, activate cytoskelatal reorganization pathways, require PI(4,5)P2 for in vitro activity and do not require Rac 1, cdc42, RhoA, PKC or ARF1 for activation.
These novel mammalian PLD proteins may be produced by recombinant genetic engineering techniques. They may also be purified from cell sources producing the enzymes naturally or upon induction with other factors. They may also be synthesized by chemical techniques, or a combination of the above-listed techniques. Mammalian PLD proteins from several species, termed PLD1a, PLD1b and PLD2, have been isolated. Human PLD1a and PLD1b are identical in amino acid sequence (SEQ ID NOS:2 and 5 respectively) except for a 38 amino acid segment that is missing from hPLD1b (SEQ ID NO:5), and present in hPLD1a (SEQ ID NO:2), beginning at amino acid number 585. Active mature PLD1a (SEQ ID NO:2) is an approximately 1074 amino acid protein, characterized by an apparent molecular weight for the mature protein of approximately 120 kD (kilodaltons) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis of protein purified from baculovirus expressing cells. The calculated molecular weight for the mature protein is approximately 124 kD. Active mature PLD1b (SEQ ID NO:5) is an approximately 1036 amino acid protein, characterized by an apparent molecular weight of approximately 120 kD as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis of protein purified from baculovirus expressing cells. The calculated molecular weight for the mature protein is approximately 120 kD. Active mature PLD2 (SEQ ID NO:8) is an approximately 932 amino acid protein, characterized by an apparent molecular weight of approximately 112 kD as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis of protein purified from baculovirus expressing cells. The calculated molecular weight for the mature protein is approximately 106 kD. As used herein, “PLD”, “PLD1a”, “PLD1b” or “PLD2” refer to any of the mammalian PLDs of this invention, “hPLD” refers to a human PLD of this invention and “mPLD” refers to a murine PLD of this invention.
Additionally, analogs of the PLD proteins and polypeptides of the invention are provided and include truncated polypeptides, e.g., mutants in which there are variations in the amino acid sequence that retain biological activity, as defined below, and preferably have a homology of at least 80%, more preferably 90%, and most preferably 95%, with the corresponding regions of the PLD1a, PLD1b or PLD2 amino acid sequences (SEQ ID NOS:2, 5 and 8 respectively). Examples include polypeptides with minor amino acid variations from the native amino acid sequences of PLD, more specifically PLD1a, PLD1b or PLD2 amino acid sequences (SEQ ID NOS:2, 5 and 8); in particular, conservative amino acid replacements. Conservative replacements are those that ta
Engebrecht Joanne
Frohman Michael A.
Morris Andrew J.
Giotta Greg
Onyx Pharmaceuticals Inc.
Saidha Tekchand
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