Cathepsin L, its prepro form and the corresponding...

Details Cathepsin L, its prepro form and the corresponding... Cathepsin L, its prepro form and the corresponding...

2001-10-18

2004-05-11

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069200, C435S069800, C435S219000, C435S252330, C435S320100, C536S023200

Reexamination Certificate

active

06733995

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the isolation of the prepro form of cathepsin L, of its leader sequence, of cathepsin L and of the affiliated propeptide from ciliates, in particular Paramecium to the use of these peptides and to a process for preparing cathepsin L from ciliates.
2. Description of the Related Art
The finding that propeptides of different proteases can, after they have been liberated by activation of the protease zymogens, act as protease inhibitors is known. For example, once splitting-off has taken place, the propeptide of
Pseudomonas aeruginosa
elastase attaches to elastase and thereby gives rise to inactivation of the enzyme (Kessler & Safrin, 1994, J. Biol. Chem., 269, 22726). The propeptides of papain and of papaya proteinase IV act selectively as inhibitors of the mature papaya proteases and of the related B and L cathepsins from rat liver (Taylor et al., 1995, Biochem. Soc. Trans., 23, 80). The propeptides of other cathepsins can also act as protease inhibitors. Thus, the synthetically prepared propeptide of human procathepsin D inhibits bovine cathepsin D (Vagner et al., 1993, Collect. Czech. Chem. Commun., 58, 435).
Cathepsin L, a protease, plays an important role in various syndromes. First, this enzyme is probably of crucial importance for the invasiveness of tumors and the formation of metastases (Pike, 1991, Dissertation Abstr. Intern., 53, 4645). This protease can also be involved in the penetration of pathogenic bacteria or parasitic protozoa into the host tissue. Cathepsin L is also involved in the degradation of bone matrix. This enzyme therefore appears to be a rewarding target in connection with the treatment of osteoporosis (Pharma Japan, September 1995, 1468, 23).
Finally, it may be mentioned that cathepsin L is also involved in the development of inflammatory diseases such as arthritis.
The identification of suitable cathepsin L inhibitors could represent an important step in the development of suitable preparations for the therapy of the said diseases. Furthermore, it would be very advantageous to have a suitable source for isolating relatively large quantities of cathepsin L. This is because the enzyme could be employed in screening systems for finding suitable protease inhibitors. Over and above this, it could be employed, for example, in wound ointments, where it could catalyze the degradation of necrotic tissue.
SUMMARY OF THE INVENTION
The present invention consequently relates to a cathepsin L prepro form which can be obtained from ciliates, preferably from Paramecium, particularly preferably from
Paramecium tetraurelia
, and to the DNA sequence encoding such a protein.
The invention furthermore relates to a cathepsin L from ciliates, preferably from Paramecium, particularly preferably from
Paramecium tetraurelia
, and the affiliated DNA sequence, to a process for its preparation from ciliates, and to its use for preparing a pharmaceutical for treating wounds.
The cathepsin L according to the present invention can furthermore be used for identifying suitable inhibitors, for example by means of so-called molecular modeling.
Furthermore, the present invention provides a cathepsin L propeptide, and its DNA sequence, from ciliates, preferably from Paramecium, particularly preferably from
Paramecium tetraurelia.
The propeptide of the cathepsin L from ciliates is a highly specific inhibitor of this cathepsin L and is consequently suitable for preparing pharmaceuticals for treating inflammatory diseases, metastasizing tumors, bacterial infections, infections with parasitic protozoa, or osteoporosis.
The present invention furthermore provides a presequence, corresponding to the leader sequence or signal ef the sequence of the cathepsin L from ciliates, preferably from Paramecium, particularly preferably from
Paramecium tetraurelia
, which presequence is translated into the corresponding leader sequence or signal sequence when recombinant peptides or proteins are expressed, thereby resulting in the secretion of the recombinantly expressed peptides or proteins from the ciliate cells.
The present invention is clarified below and with the aid of examples.


REFERENCES:
patent: 6312935 (2001-11-01), Kiy et al.
Suzuki, K.M., et al., 1998, “Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily”, European Journal of Biochemistry, vol. 254, pp. 6-13.*
Seffernick et al., 2001, “Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different”, Journal of Biochemistry, vol. 183, pp. 2405-2410.*
H. Völkel et al., European Journal of Biochemistry, vol. 238, pp. 198-206, 1996.
A. Kok and R. Paeste, Experimental Cell Research, vol. 139, No. 1, pp. 159-169, May 1982.
E. Kessler and M. Safrin, Journal of Biological Chemistry, vol. 269, No. 36, pp. 22726-22731.

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