Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-12-08
2001-04-24
Swartz, Rodney P. (Department: 1645)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C424S130100, C424S139100, C424S141100, C424S146100, C424S152100, C424S158100, C435S007400, C435S193000, C536S023200, C536S023500
Reexamination Certificate
active
06221676
ABSTRACT:
BACKGROUND OF THE INVENTION
Leukotrienes are lipid-derived cell mediators that are released in response to a variety of immunologic and inflammatory stimuli. They are products of arachidonic acid metabolism derived through the 5-lipoxygenase pathway. Briefly, the initial step in leukotriene production involves oxygenation of arachidonic acid to produce 5S-hydroperoxy-6,8-trans-11,14 cis-eicosatetraenoic acid (5-HPETE), a subsequent dehydrase step producing the epoxide intermediate, 5,6-trans-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA
4
). Two routes of metabolism from LTA
4
lead to the production of biologically active products. One of these pathways involves conjugation of LTA
4
with glutathione (GSH) via LTC
4
synthase to produce the sulfur-containing leukotriene 5S-hydroxy-6R-S-glutathionyl-7,9-trans-11,14 cis-eicosatetraenoic acid (LTC
4
). It is generally believed that LTC
4
synthase is a member of the glutathione-S transferase enzyme family.
LTC
4
has been implicated in a wide variety of diseases and pathologic conditions. LTC
4
has been identified in fluids from psoriatic lesions and bronchial secretions associated with adult respiratory distress syndrome and neonatal pulmonary hypertension. For review, see Lewis et al., New England J. Med., 323: 645 (1990), incorporated herein by reference.
Although the other enzymatic members of the 5-lipoxygenase pathway have been cloned, the cloning of LTC
4
synthase has been problematic. This is partly because the synthase is very labile in partially purified form and because the endogenous production of LTC
4
synthase in normal human cells is extremely small. LTC
4
synthase is present only in limited types of normal human cells, namely granulocytes derived from bone marrow. Moreover, oligonucleotides developed from the N-terminal region of the LTC
4
synthase polypeptide have not been specific enough to develop an effective screen because the N-terminal region is highly degenerate. In addition, an effective immunoassay for LTC
4
which relies on incubation of substrate has also been problematic since breakdown products of the substrate have been shown to cross-react with antibodies used in the assay.
It has already been established that inhibitors of 5-lipoxygenase and of the cell receptors for leukotrienes are of substantial efficacy in the management of patients with bronchial asthma. Given that that are only three points at which the leukotriene metabolic system can be disrupted: the activation and function of 5-lipoxygenase; the receptor for the leukotriene; or the function of LTC
4
synthase; characterization of LTC
4
synthase would be important, notwithstanding the problems associated with its cloning.
SUMMARY OF THE INVENTION
Human leukotriene C
4
synthase (also referred to herein as “LTC
4
synthase”) has been cloned in an expression cloning system using a highly sensitive assay for LTC
4
, the product of the reaction catalyzed by LTC
4
synthase. According to one aspect of the invention, an isolated nucleotide sequence encoding a human leukotriene C
4
synthase polypeptide or unique fragments of human leukotriene C
4
synthase polypeptide, is provided. One embodiment is an isolated DNA sequence encoding a human leukotriene C
4
synthase polypeptide that has three hydrophobic transmembrane domains. Additionally, the invention relates to mammalian leukotriene C
4
synthase nucleotide sequences isolated from murine, porcine, ovine, bovine, feline, equine, or canine, as well as primate (e.g. simian) sources.
Also provided are recombinant cells and plasmids containing the foregoing isolated DNA, preferably linked to a promoter. Portions of the foregoing nucleotide sequences are also included in the invention. One such portion is contained in a vector within a host cell.
According to another aspect of the invention, isolated human leukotriene C
4
synthase polypetide is provided, having three hydrophobic transmembrane domains. Portions of the foregoing isolated human leukotriene C
4
synthase polypeptides are also included in the invention. Antibodies with selective binding specificity for the polypeptides of the invention also are provided.
Another aspect of the invention is a method for producing human leukotriene C
4
synthase polypeptide. The method includes providing an expression vector to a host, the vector containing a DNA sequence of the invention encoding for human leukotriene C
4
synthase polypeptide, allowing the host to express the human leukotriene C
4
synthase polypeptide, and isolating the expressed polypeptide.
A further aspect of the invention is an isolated nucleotide sequence capable of hybridizing to a target nucleotide sequence encoding human leukotriene C
4
synthase polypeptide. The target includes a nucleotide sequence encoding a human leukotriene C
4
synthase polypetide with three transmembrane domains. The nucleotide sequence also can encode a human leukotriene C
4
synthase polypeptide having amino acid sequences unique to the polypeptide.
The novel molecules of the invention can be employed in experimental or therapeutic protocols. For example, a method for interfering with the activity of a human leukotriene C
4
synthase gene is provided, in which a construct is arranged to include a human leukotriene C
4
synthase nucleotide sequence that, when inserted into the genome of a cell, either inactivates transcription of messenger RNA for human leukotriene C
4
synthase polypeptide and/or inactivates translation of messenger RNA into human leukotriene C
4
synthase polypeptide in that cell. This construct further has a promotor operatively liked to the leukotriene C
4
sequence. Next, the construct is introduced into a cell, and the construct is allowed to recombine with complementary sequences of the cell genome. Finally, cells lacking the ability to express human leukotriene C
4
synthase polypeptide are selected.
A further aspect of the invention is an assay method for identifying a modulator of a human leukotriene C
4
synthase polypeptide. The method includes providing a target cell containing an isolated nucleotide sequence which encodes for a human leukotriene C
4
synthase polypeptide. The target cell is maintained under conditions and for a time sufficient for the synthase to be expressed in the target cell. The target cell is then exposed to a compound suspected of modulating human leukotriene C
4
synthase polypeptide activity and a property of the target cell is measured in the presence of the modulator. This property is also measured in an identical target cell in the absence of the modulator. An altered property of the target cell exposed to the modulator is indicative of a modulating effect of the compound.
A highly sensitive assay for LTC
4
, the product of the reaction catalyzed by LTC
4
synthase, is also described which includes the steps of contacting a carrier having bound to it an amount of an LTC
4
analogue (e.g., LTC
2
) and incubating the carrier in the presence of a solution containing an unknown amount of leukotriene C
4
synthase. Next, the carrier and solution are contacted with an amount of anti-leukotriene C
4
antibody under conditions and for a time sufficient for the anti-leukotriene C
4
antibody to bind with leukotriene C
4
in solution and with analogue (LTC
2
) on the carrier. Unbound anti-leukotriene C
4
antibody is separated from the carrier and then the carrier is contacted with a second antibody linked to a fluorescent label under conditions and for a time sufficient for the second antibody to bind with anti-leukotriene C
4
antibody associated with the carrier. The unbound second antibody is separated from the carrier and cell surface fluorescence of the carrier is analyzed by flow cytometry.
REFERENCES:
Lewis et al, “leukotrienes and other products of the 5-lipoxygenase pathway”, N.Eng. J. Med., vol. 323, No. 10, pp. 645-655, Jan. 1, 1990.*
Campbell, “General properties and applications of monoclonal antibodies”, in, Monoclonal Antibody Technology. Elsevier Science Publishers B.V., The Netherlands, pp. 1-3 and 29, Jan. 1, 1984.*
M
Austen K. Frank
Lam Bing K.
Penrose John F.
Brigham and Women's Hospital
Choate Hall & Stewart
Herschbach Jarrell Brenda
Mah Stanley C.
Swartz Rodney P.
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