Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Patent
1995-03-27
1998-03-31
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C07K 700
Patent
active
057340145
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to novel elafin derivatives, and more specifically to elafin derivatives resistant to oxidation and having similar elastase inhibiting activity to natural elafin.
BACKGROUND ART
By Wiedow et al., elafin has been isolated from psoriatics as a substance having elastase inhibiting activity and its amino acid sequence has been 3356(1991); Japanese Patent Laid-Open No. 148299/1991!. Successful chemical synthesis of this elafin (hereinafter called "natural elafin") has also been reported (The 29th Peptide Chemistry Forum, 1991).
As a result of research by the present inventors, it has however been found that natural elafin is unstable to oxidation and its activity is significantly lowered by oxidation. In particular, elastase is secreted by neutrophils, and causes damage to tissue and at the same time, produces active oxygen in abundance. To use natural elafin as an elastase inhibitor, natural elafin is strongly desired to have stability to oxidation.
DISCLOSURE OF THE INVENTION
The present inventors have conducted extensive research with a view toward obtaining an elafin derivative stable to oxidation. As a result, it has been found that its stability to oxidation is improved by partly modifying the amino acid sequence of natural elafin, leading to the completion of the present invention.
The present invention therefore provides an elafin derivative represented by an amino acid sequence of the following formula (I) (SEQ ID NO:1): ##STR2## wherein X represents leucine, isoleucine or valine, or represented by an amino acid sequence having homology to the first-mentioned amino acid sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing results of reverse phase HPLC of elafin in a reduced form and elafin in an activated form; FIG. 2 is a diagram depicting the construction of a vector capable of expressing an elafin according to the present invention; FIG. 3 is a diagram illustrating variations in neutrophilic elastase activity when non-oxidized and oxidized elafins were caused to act as inhibitors, respectively; FIG. 4 is a diagram showing variations in pancreatic elastase activity when elafins in a non-oxidized or oxidized form were caused to act, respectively; and FIG. 5 is a diagram illustrating influence of an elafin derivative according to the present invention, natural elafin and urinastatin on the survival rates of mice administered with endotoxin.
BEST MODE FOR CARRYING OUT THE INVENTION
Although the elafin derivative according to the present invention may be chemically synthesized in accordance with the formula (I) by a known peptide synthesis process, it can be efficiently prepared by causing site-directed mutagenesis on a base, which codes a predetermined amino acid of a cloning vector in which a gene coding natural elafin has been incorporated, incorporating the thus-mutated gene or a gene of the elafin derivative, said gene having been separately prepared by chemical synthesis or the like, in an expression vector, and then culturing a host microorganism which has been transformed by the expression vector.
To prepare the elafin derivative according to the present invention by site-directed mutagenesis, it is first desired to conduct cloning of the gene of natural elafin.
Since a cloning method of natural elafin is known, the cloning can be conducted following this method (Japanese Patent Laid-Open No. 148299/1991 and the like).
Next, mutation is introduced into a base sequence (hereinafter called "the predetermined base sequence") of the resulting cloning vector, said predetermined base sequence corresponding to the 25th amino acid (methionine) of natural elafin.
Illustrative methods for the introduction of this mutation include a method in which the gene of natural elafin is partly removed by a restriction enzyme or the like and an oligonucleotide separately prepared by chemical synthesis is introduced into the removed part to obtain a cloning vector modified only in the predetermined base sequence (cassette mutagenesis) and a method
REFERENCES:
patent: 5223483 (1993-06-01), Thomas et al.
patent: 5464822 (1995-11-01), Christophers et al.
Journal of Biological Chemistry, vol. 265, No. 25, pp. 14791-14795, Sep. 5, 1990, Oliver Wiedow, et al., "Elafin: an Elastase-Specific Inhibitor of Human Skin".
Proceedings of the National Academy of Sciences of USA, vol. 83, Sep. 1996, pp. 6692-6696, Robert C. Thompson, et al., "Isolation, Properties, and Complete Amino Acid Sequence of Human Secretory Leukocyte Protease Inhibitor, A Potent Inhibitor of Leukocyte Elastase".
Journal of Biological Chemistry, vol. 263, No. 15, pp. 7364-6369, May 25, 1988, Jan Potempa, et al., "An Elastase Inhibitor From Equine Leukocyte Cytosol Belongs to the Serpin Superfamily".
Biochemical and Biophysical Research Communications, vol. 166, No. 2, pp. 993-1000, Jan. 30, 1990, Mark S. Baker, et al., "Plasminogen Activator Inhibitor 2 (PAI-S) is not Inactivated by Exposure to Oxidants which can be Released from Activated Neutrophils".
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Wiedow et al., Elafin: An Elastase-specific Inhibitor of Human Skin, J. of Biol. Chem., vol. 265, No. 26, pp. 14,791-14,795, (1990).
Tsunemi et al., "Synthesis and Structure-Activity Relationships of Elatin . . . Biochem. and Biophys. Res. Com, vol. 185 No. 3, pp. 967-973, Jun. 30, 1992.
Saheki et al., "Primary Structure of the Human Elatin Precursor . . . Biochem. and Biophys Res. Com., vol. 185 No. 1, pp. 240-245, May 29, 1992.
Amagaya Sakae
Ishima Yoshiaki
Kaji Akira
Okawa Noriyuki
Yoshida Masaya
Delacroix-Muirheid C.
Tsang Cecilia J.
Tsumura & Co.
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