Platelet activating factor acetylhydrolase, and gene thereof

Details Platelet activating factor acetylhydrolase, and gene thereof Platelet activating factor acetylhydrolase, and gene thereof

1998-11-20

2001-11-27

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S183000, C435S198000

Reexamination Certificate

active

06323017

ABSTRACT:

BACKGROUND OF THE INVENTION
a) Field of the Invention
This invention relates to a novel platelet activating factor acetylhydrolase, and a gene encoding the same.
b) Description of the Related Art
A platelet activating factor acetylhydrolase is an enzyme, which acts on a platelet activating factor (hereinafter abbreviated as “PAF”) and eliminates its 2-acetyl group to deprive PAF of its activity. Since PAF is a mediator for inflammation which causes defluxion of tissue fluid through finer vessels, vasodilation, smooth muscle contraction, endothelial adhesion, activation of neutrophils, macrophages or eosinophilic leukocytes, or the like, PAF acetylhydrolase is usable as a preventive or therapeutic for various diseases caused by PAF.
Some reports have been made about PAF acetylhydrolase to date. For its use as a medicine, however, there is an outstanding desire for the provision of a PAF acetylhydrolase having higher purity and stronger action compared with conventional PAF acetylhydrolase. Further, from the viewpoint of safety, PAF acetylhydrolase derived form human being instead of an animal is desired.
SUMMARY OF THE INVENTION
With the foregoing in view, the present invention has as a primary object the provision of PAF acetylhydrolase which can fulfill the above-described desires.
Interested in the wide-spread distribution of PAF acetylhydrolase in animal organs such as the brain and kidneys, the present inventors chose the bovine liver as a source, and by various isolation and purification procedures, progressively increased the purity of PAF acetylhydrolase while placing a focus on its enzymatic activity. As a result, the present inventors have succeeded in obtaining bovine PAF acetylhydrolase as a pure product and further in determining its amino acid sequence. In addition, from the amino acid sequence of the PAF acetylhydrolase, a gene encoding the enzyme has been found by methods known per se in the art.
Moreover, using the bovine PAF acetylhydrolase cDNA, the present inventors have also succeeded in identifying the human PAF acetylhydrolase cDNA.
The present invention has been completed based on these findings, and provides a human PAF acetylhydrolase, which plays an important role as a PAF-inhibiting substance, and also a gene which encodes the enzyme and is important for the synthesis of the enzyme by genetic engineering.
The human PAF acetylhydrolase according to the present invention selectively degrades PAF and hence, is usable as medicines or biochemical reagents for the prevention and treatment of diseases caused by PAF, for example, diseases such as asthma, exudative tympanitis, hemorrhagic colitis and adult respiratory distress syndrome.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
The human PAF acetylhydrolase according to the present invention can be prepared as will be described next. PAF acetylhydrolase is first collected from an animal. From the PAF acetylhydrolase, the animal PAF acetylhydrolase cDNA is determined. Using the animal PAF acetylhydrolase cDNA, the human PAF acetylhydrolase cDNA is detected from a human gene library. The human PAF acetylhydrolase cDNA is inserted in an appropriate vector and then cultures in an adequate host organism, whereby the human PAF acetylhydrolase is obtained.
Upon practice of the present invention, it is first necessary to obtain animal PAF acetylhydrolase from an organ of an animal such as the brain, liver or kidneys by purifying it through repetitions of known isolation and purification procedures while using PAF acetylhydrolase activity as an index. A description will hereinafter be made of a process for obtaining PAF acetylhydrolase by using a bovine liver as an example.
As the bovine liver to be used as a source, one obtained from a bovine immediately after its slaughter is preferred.
After the bovine liver is first washed with an appropriate buffer (for example, 10 mM Tris-HCl buffer containing 250 mM sucrose and 1 mM EDTA and having a pH of 7.4), it is homogenized with the same buffer. The homogenate is then centrifuged to obtain a soluble fraction.
Making combined use of hydrophobic chromatography, ion exchange chromatography, adsorption chromatography, gel filtration chromatography and the like, the soluble fraction is purified until a single peak is observed by Mono Q FPLC, so that PAF acetyl hydrolase can be obtained.
Incidentally, PAF acetylhydrolase activity which is used as an index for the selective collection of the PAF-acetylhydrolase-containing fraction can be determined, for example, by the method disclosed in Japanese Patent Application Laid-Open (Kokai) No. HEI 7-39373.
With respect to the bovine PAF acetylhydrolase obtained in the above-described manner, its amino acid sequence was investigated by a method known per se in the art. As a result, the amino acid sequence has been found to be represented by the following formula (III) (SEQ ID NO:1):
Met Gly Val Asn Gln Ser Val Ser Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Ser Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala Gly Leu Ala Glu Tyr Leu Lys Phe Asn Lys Arg Trp Gly Gly Leu Leu Phe Asn Leu Gly Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Met Gly Ala Phe Arg Thr Val Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr Cys Phe Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro Asp Asn Glu Ala Leu Lys Glu Glu Trp Ile Pro His Arg Gln Ile Glu Glu Gly Glu Lys Glu Phe Tyr Val Arg Asn Tyr Gln Val His Gln Arg Val Ser Glu Cys Val Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Ala Val Leu Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Gly Ile Asp Val Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu Ala Lys Glu Met Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro Leu Glu His Asp Phe Tyr Pro Thr Ala Arg Gly Pro Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val Glu Thr Val Asn Leu Met Lys Lys Ile Cys Asp Gln His His Gln Ser Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Leu Thr Asp Phe Val Phe Val Ala Gly Asn Trp Ile Ser Lys Phe Phe Ser Ser His Thr Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr Val Val Arg Ala Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asp Gln Trp Asn Ash Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser Ser Leu (III)
Further, from the peptide sequence of the bovine PAF acetylhydrolase of the formula (III), a gene encoding the enzyme was determined by a method known per se in the art. The gene (hereinafter called the “bovine PAF acetylhydrolase cDNA”) has been found to be identified by the following formula (IV) (SEQ ID NO:2):
GTCGACCCACGCGTCCGAGTTGACCGT CTGGGCTGTTTCTGAGGGTCAACGTGACTCGCCGTCAAGTTCAGCCACTGCCCAAGTCGT CGTTCAGTTCAGTTGGTTATGAG ATG GGG GTC AAC CAG TCT GTG AGC TTC CCA CCC GTC ACG GGA CCC CAC CTC GTA GGC TGT GGG GAT GTG ATG GAG GGT CAG AGC CTC CAG GGC AGC TTC TTT CGA CTG TTC TAC CCG TGC CAA GAG GCA GAG GAG ACC TCG GAG CAG CCC CTG TGG ATT CCC CGC TAT GAG TAC TGC GCT GGC CTG GCC GAA TAC CTA AAG TTT AAT AAG CGC TGG GGG GGG TTA CTG TTC AAC CTG GGT GTG GGA TCT TGT CGC CTG CCT GTT AGC TGG AAT GGC CCC TTT AAA ACA AAG GAC TCT GGA TAC CCC TTG ATC ATC TTC TCT CAT GGC ATG GGA GCC TTC AGG ACA GTG TAT TCA GCC TTC TGC ATG GAG CTG GCT TCT CGT GGC TTT GTG GTT GCT GTA CCA GAG CAC AGG GAT GGG TCA GCT GCG GCC ACC TGT TTC TGC AAG CAG ACC CCA GAG GAG AAC CAG CCT GAC AAT GAG GCC CTG AAG GAG GAA TGG ATC CCC CAC CGT CAA ATT GAG GAA GGG GAG AAG GAA TTC TAT GTT CGG AAC TAC CAG GTG CAT CAG AGG GTG AGC GAG TGT GTG AGG GTG TTG AAG ATC CTA CAA GAG GTC ACT GCT GGG CAG GCC GTT CTC AAC ATC TTG CCT GGC GGA TTG GAT CTG ATG ACC TTG AAG GGC GGC ATT GAC GTG AGC CGT GTG GCT GTA ATG GGA CAT TCA TTT GGA GGG GCC ACA GCT ATT CTG GCC TTG GCC AAG GAG ATG CAA TTT AGG TGT GCT GTG GCT TTG GAC GCT TGG ATG TTT CCT CTG GAG CAT GAC TTT TAC CCC ACG GCC CGA GGC CCT ATC TTC TTT ATC AAT GCT GAG AAG TTC CAG ACA GTG GAG ACT GTC AAC TTG ATG AAA AAG ATT TG

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