Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-03-06
2001-04-10
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06214599
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an enzyme having a new cysteine protease-like activity. The enzyme of this invention is a bimolecular protease comprising two subunits, that is, one subunit having a molecular weight of about 26 kDa and the other having a molecular weight of about 29 kDa.
2. Prior Art
Proteases are mainly classified into four categories including serine proteases, cysteine proteases, aspartic acid proteases and metalloproteases according to their structure and catalytic activity. Among them, the proteases which include the most numerous members and have been studied most intensively from old times are serine proteases and cysteine proteases. The serine protease includes the majority of digestive proteases and serum proteases, and attention has been paid to their extracellular functions thereof. In contrast, the cysteine protease has been believed to be an important group of enzymes in the intracellular digestion of proteins (Katsunuma, N., “Intracellular digestion of proteins (in Japanese),” pp. 35-50, Tokyo Kagaku-Dojin Publishing Co.).
As representative examples of the cysteine protease, the cathepsin type proteases such as cathepsin L and cathepsin B may be mentioned, and these enzymes have been assumed to be lysosomal proteases. The lysosomal protease is a protease which digests, in the cell, proteins carried into lysosomes or one of intracellular organellae, and is believed, on the basis of its basic role in biological functions, to exist universally in organisms.
Cathepsin L and cathepsin B were also demonstrated in a flesh fly (Sarcophaga peregrina) which undergoes a complete metamorphosis (Kurata, S., et al. (1982) Eur. J. Biochem., 204, 911-914; Takahashi, N., et al. (1933) FBBS. Lett. 334, 153-157; Homma, K., et al. (1994) J. Biol. Chem., 269, 15258-15264). Cathepsin L and cathepsin B demonstrated in the fly, so it was found, not only act as a lysosome enzyme but also function as a secretory enzyme involved in morphogenesis in the manner as described below. Namely, cathepsin B of flesh fly, while the fly is developing from a third-instar larva into an adult, is discharged from body fluid cells and digests unneeded larval tissues and fat bodies discharged from the body fluid (Kurata, S., et al. (1989) J. Insect Physiol. 35, 559-565; Kurata S., et al. (1990) Insect Biochem. 20, 461-465; Kurata, S., et al. (1992) Dev. Biol., 153, 115-121).
Further, cathepsin L of flesh fly is discharged from an imaginal disk when stimulated with ecdysone, and digests the basal membrane of the imaginal disk to help the imaginal disk to develop into the matured structure of the imaginal disk (Homma, K., et al. (1994) J. Biol. Chem. 269, 15258-15264).
As described above, it was discovered that cathepsin L and cathepsin B, which had been originally assumed to be a lysosome enzyme, has a novel property hitherto unknown, that is, they function also extracellularly in certain aspects of insect life.
From the flesh fly, another secretory cysteine protease, that is, 26·29 kDa protease was newly discovered. This 26·29 kDa protease is a protease isolated from the body fluid cells of a flesh fly and purified, and as its activity is inhibited by E64 or a specific inhibitor of cysteine proteases, it has been assumed that the protease in question is one of cysteine proteases. However, in contrast with known cysteine proteases, the protease in question has following new properties.
(1) It has a unique structure, that is, it comprises two subunits having molecular weights of 26 kDa and 29 kDa, respectively.
(2) The protease, so it was demonstrated, is discharged from body fluid cells into the body fluid when sheep red cells are injected as a foreign substance into the body of a third-instar larva of the flesh fly. From this fact, it has been assumed that this protease serves for the body protection of the larva.
Regarding cysteine proteases, various molecular variants have been reported heretofore, but the majority of them have a property of a monomolecularity, and the 26·29 kDa protease was thought possibly a new protease hitherto unknown, because it is a bimolecular type protease. The 26·29 kDa protease never fails to exist as a bimolecule type whenever it was found to have an enzymatic activity, and thus it was assumed that this substance may be a protease advanced in evolution one step ahead of fellow proteases which, comprising two subunits to act in a concerted manner, may execute a new function inaccessible to monomolecular fellow proteases.
Furthermore, there have been no known cysteine proteases that are secreted in response to a nocuous stimulus such as injection of a foreign substance like the one as described in (2) above, and in this respect too, it was assumed that the 26·29 kDa protease is a new cysteine protease.
SUMMARY OF THE INVENTION
This invention relates to a new protease, or the 26·29 kDa protease.
Namely, this invention relates to proteins having amino acid sequences as represented by the attached Sequence Listing, SEQ. ID. NOS. 1, 3 and 5, and having a cysteine protease-like activity.
Further, this invention relates to a DNA sequence having a base sequence as represented by the attached Sequence Listing, SEQ. ID. NOS. 2, 4 or 6.
Furthermore, this invention relates to an enzyme comprising a protein having an amino acid sequence as represented by the attached Sequence Listing, SEQ. ID. NOS. 1, 3 or 5, and having a cysteine protease-like activity.
REFERENCES:
Eur. J. Biochem. 209, 939-944, 1992.
Saidha Tekchand
Schneller Marina V.
University of Tokyo
Venable
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