Phosphatidylcholine phospholipase D

Details Phosphatidylcholine phospholipase D Phosphatidylcholine phospholipase D

1998-06-30

2001-08-14

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S252300, C435S320100, C536S023200, C536S023500

Reexamination Certificate

active

06274363

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
In general, the invention pertains to human polynucleotide sequences encoding for polypeptides having enzymatic activity relevant in cell signaling. The present application pertains in particular to mammalian phosphatidylcholine phospholipase D (PCPLD), specifically, human PCPLD (hPCPLD), to fragments and polypeptide analogs thereof and to polynucleotides encoding the same.
BACKGROUND OF THE INVENTION
Cell activation is associated with rapid upregulation of synthesis of phospholipids (PL) that includes phosphatidic acid (PA), diacylglycerol (DAG) and phosphatidylinositol (PI). PA's are a molecularly diverse group of phospholipid second messengers coupled to cellular activation and mitogenesis. Singer et al.,
Exp. Opin. Invest. Drugs
3:631643, 1994. Compounds capable of modulating PA generation and hence altering a signal involved in cell activation may therefore be of therapeutic interest in the area of inflammation and oncology.
Lysophosphatidic acid acyltransferase (LPAAT) is an important enzyme in the synthesis of a specific species of PA in activated monocytic cells. Rice et al.,
Proc. Nat'l Acad. Sci. USA
91:3857-3861, 1994. PCPLD is another major enzyme class involved in the generation of PA through hydrolysis of phosphatidyl choline (PC) into PA and choline. Exton,
Biochim Biophys Acta
1212:2642, 1994). Okamura et al.,
J. Biol. Chem.
269:31207-31213, 1994, report PCPLD protein purification from pig lung. Brown et al.,
J. Biol. Chem.
270:14935-14943, 1995, report PCPLD protein purification from porcine brain, and Vinggaard et al. discuss PCPLD isolation from human placenta.
Biochim Biophys Acta
1258:169-176, 1995.
In plant species, Wang et al. published results of cloning efforts with castor bean PCPLDs.
J. Biol. Chem.
269:20312-20317, 1994. Ueki et al. disclose PCPLD purified from rice and maize,
Plant Cell Physiol.
36:903-914, 1995, and there also are reports on PCPLD isolation and purification from yeast. Ella et al.,
Biochem. J.
314, 15-19, 1996; Rose et al.,
Proc. Natl. Acad. Sci.
92: 12151-12155, 1995.
Most recently, Hammond et al. report cloning of a human isoform of PCPLD, hPLD1.
J. Biol. Chem.
270: 29640-29643, 1995. SEQ ID NO. 3 is a sequence listing of the amino acids of hPLD1. Based on a variety of biochemical studies including differential subcellular fractionation, distinct mechanism of activation, substrate specificity and different chromatographic properties, evidence for the existence of multiple phospholipase D (PLD) isoforms in mammalian cells is growing rapidly. Liscovitch et al.,
Chem. Phys. Lipids
80: 37-44, 1996; Kiss,
Chem. Phys. Lipids
80: 81-102. hPLD1 has approximately a 40% sequence homology with hPCPLD.
Although other mammalian PLD sequences have been cloned, heretofore the sequence of the disclosed PCPLD has not been obtained. Therefore, cloning cDNA isoforms of PLD that are closely related to other mammalian and plant isoforms of PLD would be useful in conducting discovery research to identify specific agents capable of modulating this enzyme.
SUMMARY OF THE INVENTION
This present invention relates to three, previously unknown isoforms, hPCPLD2.1, hPCPLD2.2, and hPCPLD1.5, which are hereafter called “hPLD2.1,” “hPLD2.2” and “HPLD1.5,” respectively. Thus, the invention provides cDNA sequences, polypeptide sequences, and transformed cells for producing isolated, recombinant hPLD2. 1, hPLD2.2 or hPLD1.5. The invention conternplates, inter alia, the incorporation of codons “preferred” for expression by selected nomnammalian hosts, the provision of sites for cleavage by restriction endonuclease enzymes, and the provision of initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors.
The invention also provides DNA sequences coding for microbial expression of polypeptide analogs or derivatives of bPLD2.1, hPLD2.2 or hPLD1.5, which differ from naturally-occurring forms, in terms of the identity or location of one or more amino acid residues, and which share some or all properties of naturally occurring forms. Accordingly, the invention encompasses deletion analogs that contain fewer than all of the residues specified for hPLD2. 1, hPCD 2.2, or hPLD1.5; substitution analogs, such as [Ser
17
]PLD, where one or more amino acid residues are added to a terminal or medial portion of the polypeptide.
As described in greater detail below, hPLD2.1 and hPLD2.2 polypeptides display PCPLD activity in a particular fluorescent assay. Accordingly, the present invention includes polynucleotide sequences that are useful for expressing, in procaryotic or eucaryotic host cells, polypeptide products that have at least a primary structure and a biological property in common with naturally-occurring hPLD2. 1 or hPLD2.2.
With the aforementioned assay, the present inventors have not observed activity associated with hPLD1.5, under circumstances where hPLD2.1 and hPLD2.2 were active in the assay (see below). Because hPLD1 and hPLDI.5 share substantial aspects of primary structure, however, hPLD1.5 may display PCPLD activity under other circumstances. In any event, the present invention encompasses assays for screening test compounds for their ability to inhibit hPLD2.1 or hPLD2.2. Accordingly, hPLD1.5 protein can be used as a negative control in the context of screening compounds for inhibition of PCPLD1 activity, pursuant to the present invention. Also, a polynucleotide encoding hPLD1.5 can be used as a probe to identify genes encoding other PCPLD isoforms.
More generally, the present invention contemplates a category of polynucleotides that _includes, without limitation, (a) an isolated DNA that encodes hPLD2.1, hPLD2.2 or hPLD1.5; (b) a DNA that hybridizes, under conditions such as are illustrated herein or are more stringent conditions, to a DNA set forth in this specification or to a fragment thereof; (c) a DNA that, but for the degeneracy of the genetic code, would hybridize to DNA sequences disclosed herein; and (d) an antisense oligonucleotide for modulating expression of hPLD2. 1, hPLD2.2 or hPLD1.5. Subcategory (b) includes, without limitation, genomic DNA sequences encoding allelic variants of hPLD2.1, hPLD2.2 or hPLD1.5. Subcategory (c) includes, without limitation, manufactured DNAs encoding hPLD2.1, hPLD2.2 or hPLD1.5, fragments of these proteins, and analogs of the proteins, which DNAs optionally incorporate codons facilitating translation messenger RNA in a prescribed microbial or other host.
To these ends, the present invention provides, in accordance with one of its aspects, a polynucleotide (i) that codes for a PCPLD isoform selected from group consisting of hPLD2. 1, hPLD2.2, and hPLD1.5 or (ii) that hybridizes to a polynucleotide encoding said isoform. In a preferred embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NOS.
1 & 2, 16 & 17
or
18 & 19,
respectively.
In accordance with another aspect of the present invention, an isolated PCPLD isoform is provided, selected from a group consisting of hPLD2. 1, hDLD2.2, and hPLD1.5. Pursuant to one preferred embodiment, the isolated PCPLD isoform comprises the amino acid sequence of SEQ ID NOS.
1 & 2
or of SEQ ID NO.
16 & 17,
or an enzymatically active fragment thereof. According to another embodiment, the isolated PCPLD isoform comprises the amino acid sequence of SEQ ID NOS.
18 & 19.
In accordance with yet another aspect of the present invention, a method is provided for screening a drug candidate, comprising (a) providing at least one of the aforementioned isoforms that displays PCPLD activity, (b) contacting that isoform with the drug candidate, and then (c) determining whether the drug candidate affects PCPLD activity of the isoform.
In a preferred embodiment, step (c) comprises measuring the PCPLD activity of the isoform against a control sample, which can contain a PCPLD isoform comprising the amino acid sequence of SEQ ID NOS.
18 & 19.
In another embodiment, the drug candidate is a pool of compounds from combinatorial library expression.

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