Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-06-11
2004-06-29
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S440000, C536S023200, C424S094600
Reexamination Certificate
active
06756219
ABSTRACT:
TECHNICAL FIELD
This invention relates to a human secretory type phospholipase A
2
; a DNA encoding this protein; a vector having the DNA; a transformant having the vector; a method for producing the protein by using the transformant; an antibody recognizing the protein; a screening method for a compound by using the protein; and a compound obtained by the screening method.
BACKGROUND ART
Phospholipase A
2
(PLA
2
; EC 3.1.1.4) is a general term of phospholipids-cleaving enzymes that hydrolyze the 2-acyl ester bond of 3-sn-phosphoglyceride. PLA
2
is involved in the digestion of phospholipids in food and the generation and metabolism of phospholipids in the cell membranes. In addition, PLA
2
plays as a rate-limiting enzyme of the arachidonic acid cascade in the production of lipid mediators including prostaglandins. It has been known that various types of PLA
2
s exist in mammals. PLA
2
s are classified into 4 different families, such as the secretory PLA
2
, cytosolic PLA
2
, Ca
2+
-independent PLA
2
, and platelet-activating factor-acetylhydrolase, based on the localization, Ca
2+
requirement, and substrate specificity (Balsinde et al., J. Biol. Chem. 272, 16069-16072 (1997)).
Among them, secretory PLA
2
family comprises PLA
2
enzymes that are secreted into the outside of the cells and have a relatively low molecular weight (13,000-15,000). As the member of the family, 5 types including IB type, IIA type, IIC type, V type, and X type, have been already identified. Each molecule has 12 to 16 Cys residues that form disulfide-bonds in the molecule, and possesses a consensus active site consisting of His-Asp residues. In addition, the molecules have a common Ca
2+
binding region. Micro mole order of Ca
2+
concentration is required for the exertion of the enzyme activity (Tischfield et al., J. Biol. Chem., 272, 17247-17250 (1997), Cupillard et al., J. Biol. Chem., 272, 15745-15752 (1997)).
It is assumed that IB type has a function as a digestive enzyme in the pancreas etc. and is involved in the progression of inflammation, such as endotoxin shock, through the binding to its specific receptor. It is also assumed that IIA type plays a role in various inflammatory responses because this type is expressed in blood platelets and synovial cells etc., and its expression is elevated during stimulation of inflammatory cytokines. However, the inflammatory response is normal in genetically IIA-deficient mice. Thus, its pathological significance remains unresolved. V type is expressed in the heat and several inflammatory cells. X type is expressed in the tissues related to the immunity such as spleen and thymus. Although it is suggested that both types are involved in the bio-regulation and inflammatory response, their importance in the body is not clearly identified (Hanasaki et al., Cell Technology, 17, 694-701 (1998)).
DISCLOSURE OF INVENTION
The object of this invention is to provide a novel type of human secretory phospholipase A
2
; a DNA encoding the protein of this invention; a vector comprising the DNA of this invention; a transformant having the vector of this invention; a method for producing human secretory type phospholipase A
2
by using the transformant of this invention; an antibody specifically recognizing the protein of this invention; a screening method of a compound by using the protein of this invention; and a compound obtained form the screening method.
In the process of intensive studies regarding the physiological function of mouse X type PLA
2
, the inventors found the presence of partial sequence with homology to mouse X type PLA
2
in Expressed Sequence Tags (EST) database. Based on the partial sequence, they found a DNA sequence encoding novel secretory type PLA
2
protein from mouse spleen cDNA library. Further, the inventors found out a DNA sequence encoding human secretory type PLA
2
protein (IID type) from human spleen cDNA library, to accomplish this invention.
The invention relates to:
A protein which comprises an amino acid sequence from First Gly to 125
th
Cys of that shown in SEQ ID No.:27;
The protein as described above which comprises an amino acid sequence from −20th Met to 125th Cys of that shown in SEQ ID No.:27;
A protein which comprises the above described amino acid sequence, in which one or more amino acid residues are substituted, deleted, inserted, or added, and has a secretory type phospholipase A
2
activity;
A DNA which encodes the protein as described above;
The DNA as described above which comprises a base sequence from 89th A to 463rd C of that shown in SEQ ID No.:26;
The DNA as described above which comprises a base sequence from 29th to 463rd C of that shown in SEQ ID No.26;
A DNA which hybridizes to the DNA as described above under the stringent condition and encodes the protein having a secretory type phospholipase A
2
activity;
A vector which has the DNA as described above;
A transformant which is obtained by inserting the expression vector as described above to a host;
The transformant as described above wherein the host is a mammalian cell line;
A method for producing recombinant secretory type phospholipase A
2
which comprises a step for culturing the transformant as described above and a step of recovering a produced recombinant protein from the culture;
An antibody which specifically recognizes the protein as described above;
A diagnostic agent for secretory type phospholipase A
2
-related diseases, which comprises the antibody as described above;
An assay kit for secretory type phospholipase A
2
, which comprises the antibody as described above;
A therapeutic agent for secretory type phospholipase A
2
-related diseases, which comprises the antibody as described above;
A screening method of a compound specifically inhibiting a secretory type phospholipase A
2
activity which uses the protein as described above; and
A compound which is obtained by the screening method as described above.
The protein of this invention is “A protein which comprises an amino acid sequence from first Gly to 125th Cys of that shown in SEQ ID No.:27”. Preferable is “A protein which comprises an amino acid sequence from −20th Met to 125th Cys of that shown in SEQ ID No.:27”. “A protein which comprises an amino acid sequence from first Gly to 125th Cys of that shown in SEQ ID No.:27” means a mature protein. “A protein which comprises an amino acid sequence from −20th Met to 125th Cys of that shown in SEQ ID No.:27” is an immature protein that has a signal peptide. The protein of this invention also includes “A protein which comprises the amino acid sequence as described above, in which one or more amino acid residues are substituted, deleted, inserted, or added, and has a secretory type phospholipase A
2
activity”. Number or site of “substitution, deletion, insertion, addition of amino acid residue” is not limited, if the modified protein has the same activity as the protein consisting of the amino acid sequence shown in SEQ ID No.:27. In this invention, “phospholipase A
2
activity” means “phospholipid-cleaving activity that hydrolyzes 2-acyl ester bond of 3-sn-phosphoglyceride in a Ca
2+
-dependent manner”.
Although these mutations in the amino acid sequences can be caused naturally by mutation or modification after transcription, artificial modification can also be caused by the DNA of this invention. The protein of this invention includes all proteins which are encoded by modified DNA have the characteristics as mentioned above regardless of the cause or mean of these modification/mutation.
The DNA of this invention means “a DNA encoding the protein of this invention”. As the DNA of this invention, a DNA encodes the mature protein and comprises a base sequence from 89th G to 463rd C of that shown in SEQ ID No.:26 is preferably given for example. More preferably, a DNA encodes the immature protein and comprises a base sequence from 29th A to 463rd C of that shown in SEQ ID No.:26 is given for example. A DNA that hybridizes to the DNA of this invention under the stringent condition and encodes the
Hanasaki Kohji
Ishizaki Jun
Suzuki Noriko
Achutamurthy Ponnathapu
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Pak Yong
Shionogi & Co. Ltd.
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