Cathepsin L, its prepro form and the corresponding propeptide from ciliates

Abstract

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.

Description

[0001] 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.

[0002] 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).

[0003] 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).

[0004] Finally, it may be mentioned that cathepsin L is also involved in the development of inflammatory diseases such as arthritis.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] Furthermore, the present invention provides a cathepsin L propeptide, and its DNA sequence, from ciliates, preferably from Paramecium, particularly preferably from Paramecium tetraurelia.

[0010] 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.

[0011] The present invention furthermore provides a presequence, corresponding to the leader sequence or signal 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.

[0012] The present invention is clarified below and with the aid of examples.

[0013] The present study describes, for the first time, the isolation of two proteases of the cathepsin L subfamily from the ciliate Paramecium (Protista). Sequencing of the cloned cDNA demonstrates that while conformity with previously described cathepsin L forms from Mammalia and Protista is at most 30%, the characteristic cathepsin L motifs are present both in the prepro region and in the actual enzyme. The pro region encodes a segment of 86 amino acids in length which exhibits the typical ERFNIN motif. The pro region was expressed in E. coli. The isolated propeptide efficiently (in the nanomolar region) inhibited the Paramecium cathepsin L. By contrast, other cysteine proteases, for example papain and mammalian B, G and H cathepsins were not inhibited even at propeptide concentrations of 13 μM. The propeptide is consequently an effective and specific inhibitor of cathepsin L. Based on these data, it should be possible to develop a potent and highly specific inhibitor for chemotherapeutic use in the treatment of the abovementioned syndromes.

EXAMPLE

[0014] Cathepsin L Assay

[0015] 32 P-Phosphorylase a (approx. 5×104 cpm/min) was used as substrate. A test mixture (30 μl) contained 10 μM substrate, 12 mM Tris/HCl (pH 7.0), 50 μM EDTA, 10 mM 2-mercaptoethanol, 5 mM caffeine and 6.7 μg of BSA. The reaction was stopped, after incubating at 30° C. for 10 minutes, by adding 200 μl of trichloroacetic acid (20% w/v). The radioactivity of the non-precipitable peptides was determined in the supernatant following centrifugation. One unit of enzyme activity corresponds to the quantity which liberates 1 μmol of soluble 32P-phosphopeptide/min.

[0016] Purification of the Cathepsin L

[0017] Mass cultures of the ciliate Paramecium tetraurelia were used as the source. Cathepsin L can be obtained both from the cells and, in large quantities, from the culture medium, since the cells also secrete the enzyme.

[0018] All the purification steps were carried out at 4° C. The cells were homogenized in 50 mM Tris/HCl (pH 7.0), 5 mM EDTA using a French press. Cell debris were removed by centrifugation (23,000× g, 60 min; 100,000× g, 60 min). The supernatant was loaded onto a DEAE Sepharose® column which was equilibrated with 20 mM Tris/HCl (pH 7.0). About half the protease activity eluted with the flowthrough. The column was washed with 250 mM NaCl. The remaining protease activity was eluted with 450 mM KCl. After that, the active fractions were purified through a Sephacryl® S-100 HR column. The protease eluted at approximately 27 kDa. The pooled active fractions were subsequently loaded onto a mono Q column. Elution was carried out using a linear gradient (60 ml of from 100 to 350 mM NaCl). Two active proteases (30 kDa and 33 kDa) were separated in this step. Purity was examined by means of SDS-PAGE. When using 32P phosphorylase a as substrate, the pH optimum of the two isozymes was 6.5; the temperature optimum was 56° C. Sulfhydryl protease-specific inhibitors (e.g. cystatin, leupeptin and TLCK) reduced the activity drastically. On the other hand, inhibitors which were specific for serine proteases (aprotinin), metalloproteases (EDTA) and Asp proteases (pepstatin) had no inhibitory effect. The digestion pattern obtained with phosphorylase and BSA indicated that the two proteases were endoproteinase isozymes.

[0019] Amino Acid Sequencing

[0020] The proteins were blotted out of the SDS gel onto a polyvinylidene difluoride membrane, and the corresponding 30 kDa and 33 kDa bands were cut out. For the sequencing of protein fragments, the proteins were cleaved with BrCN (350 μg{10 μg of protein) prior to the SDS-PAGE. The sequencing was carried out on an Applied Biosystems sequencer. The NH2 terminus of the 30 kDa band is: GAEVDWTDNKKVKYPAVKNQ, while that of the 33 kDa band is: GAEVDXTXNK (X is unresolved). The sequencing of the BrCN fragments also showed that identical enzyme proteins were involved, with the proteins possibly only being processed differently. In this case, the following sequence was determined for both the proteins: DSAFEYVADNGLAEAKDYPYYASD. Comparison with the EMBL gene bank using the FASTA program did not indicate any correspondence with known proteins as far as the NH2 terminus was concerned; on the other hand, alignment of the internal 24mer peptide demonstrated unambiguous correspondence with 19 different cysteine proteases.

[0021] Amplification and Subcloning of Cathepsin L

[0022] Oligonucleotides were prepared on the basis of the amino acid sequencing and taking into account ciliate codon usage. The primers employed were: primer 1 (sense) 5′-GCGGGGTACCGGWGCHGAAGTHGAYTGGACWGA-TAAYAARAARG-3′, deduced from the NH2-terminal peptide GAEVDWDNKKVK and primer 2 (antisense) 5′-TARTANGGRTARTCYTTNGCYTC-3′, deduced from the internal peptide sequence EAKDYPYY. The PCR was carried out in a Perkin-Elmer Thermal Cycler (30 cycles, at 94° C., 55° C. and 72° C. for 1 min in each case). Using these primers, a fragment of 275 bp in length was amplified from a Paramecium cDNA library. Sequencing this DNA fragment provided unambiguous evidence of its similarity to cathepsin L. Thus, the PCR fragment contained the two strongly conserved regions GCNGG and CGCSWA. Two clones having inserts of 1.3 kB were identified in the cDNA library using the 275 bp fragment. Sequencing these clones indicated that they contained identical open reading frames which encoded a protein of 313 amino acids having a calculated molecular weight of 35,031 Da (FIG. 2). The deduced amino acid sequence was in agreement with that determined by means of Edman degradation.

[0023] The conserved ERFNIN motif in the propeptide (EX3RX2(V/I)FX2NX3IX3N) characterizes the enzyme as H or L cathepsin. Whereas cathepsin H is characterized as an exoprotease, cathepsin L is classified as an efficient endoprotease. The identification as endoproteases of the proteases which are described here suggests that they are in fact forms of cathepsin L. The correspondence of the Paramecium cathepsin L to different mammalian forms is at most 35% (Tab. 1). The correspondence is also only 30% when compared with the Tetrahymena cysteine protease.

TABLE 1
Cathepsins and proteases as	% identity with
compared with	SWISSPROT	mature
Paramecium cathepsin L	accession No.	proteases	pro regions
Rat L type	P07154	35	21
Tetrahymena cysteine	L03212	30	23
protease
Rat H type	P00786	30	19
Rat S type	Q02765	31	19
Human B type	P07858	21	12

[0024] cDNA Library Screening

[0025] 32 P-labeled PCR fragments were used to screen the cDNA library for corresponding clones. The two clones which were identified in this way were analyzed by Southern blotting. Both the clones encoded an identical preprocathepsin L protease.

[0026] Bacterial Expression of the Cathepsin L Propeptide

[0027] The cloned gene contains a potential propeptide region from AA −1 to −86. The open reading frame contains five universal TAA stop codons, which encode Q in Paramecium. Before being expressed, they were changed into CAA (encodes Q) by means of site-directed mutagenesis.

[0028] The DNA fragment containing the propeptide region was amplified by PCR and introduced, for expression, into the heat-inducible vector pEV41 C, which additionally contained a hexa-His tag. The primers which were used for the PCR were 5′-AGGTCGTCATATGAATCTTTATGCAAATTGG (sense) and 5′-ATCCTCGAGTCACTTGTATTGGAAGTTAG (antisense). Following transformation, the propeptide was expressed in E. coli strain 2136. Expression was induced by adding LBsmp medium which had been preheated to 42° C.

[0029] After harvesting, the cells were homogenized and the cell debris was removed by centrifugation. The supernatant was purified on an Ni affinity column (Qiagen). The protein was eluted using 20 mM Tris/HCl (pH 7.5), 8,6% glycerol, 200 mM NaCl and 500 mM imidazole. As expected, a protein having a size of 13.6 kDa was eluted under these conditions.

[0030] In an inhibition test, the propeptide inhibited the 30 kDa cathepsin L isozyme from Paramecium by 50% at a concentration of only 60 nM (FIG. 1). Other proteases (papain, human liver cathepsin H, bovine kidney cathepsin B and leukocyte cathepsin G) were not inhibited even at propeptide concentrations of 13 μM.

Claims

1. A prepro form of cathepsin L, obtainable from ciliates.

2. A prepro protein as claimed in claim 1, wherein the ciliate is Paramecium.

3. A prepro protein as claimed in claim 2, wherein the ciliate is Paramecium tetraurelia.

4. A DNA sequence encoding a prepro form of cathepsin L as claimed in one of claims 1 to 2.

5. The DNA sequence shown in FIG. 2.

6. The antisense strand of the DNA sequence shown in FIG. 2.

7. The amino acid sequence shown in FIG. 2.

8. The presequence of the DNA sequence as claimed in claim 4.

9. The leader sequence from the prepro form of cathepsin L as claimed in one of claims 1 to 2.

10. The presequence corresponding to bases Nos. 1 to 86 of the DNA sequence shown in FIG. 2.

11. The antisense strand of the sequence as claimed in claim 10.

12. The coding presequence of the sequence as claimed in claim 10, corresponding to bases Nos. 21 to 86 of the DNA sequence shown in FIG. 2.

13. A DNA sequence as claimed in claim 12, wherein all the TAA codons are replaced with those codons which encode Q in the corresponding expression system.

14. A DNA sequence as claimed in claim 13, wherein all the TAA codons are replaced with CAA codons.

15. An antisense strand of the sequence as claimed in one of claim 12 or 14.

16. The amino acid sequence corresponding to amino acids Nos. −108 to −87.

17. The pro region of the DNA sequence as claimed in claim 4.

18. The propeptide contained in the prepro form of cathepsin L as claimed in one of claims 1 to 2.

19. The pro region of the DNA sequence shown in FIG. 2, corresponding to bases Nos. 87 to 347.

20. A pro region as claimed in claim 19, wherein all the TAA codons are replaced with those codons which encode Q in the corresponding expression system.

21. A pro region as claimed in claim 20, wherein all the TAA codons are replaced with CAA codons.

22. An antisense strand as claimed in one of claim 19 or 21.

23. The propeptide of the amino acid sequence shown in FIG. 2, corresponding to amino acids Nos. −86 to −1.

24. A cathepsin L, obtainable from ciliates.

25. A cathepsin L as claimed in claim 24, wherein the ciliate is Paramecium.

26. A cathepsin L as claimed in claim 25, wherein the ciliate is Paramecuim tetraurelia.

27. The DNA sequence corresponding to bases Nos. 348 to 1276 in FIG. 2.

28. The coding DNA sequence, corresponding to bases Nos. 348 to 965 in FIG. 2.

29. A DNA sequence as claimed in claim 28, wherein all the TAA codons are replaced with those codons which encode Q in the corresponding expression system.

30. A DNA sequence as claimed in claim 29, wherein all the TAA codons are replaced with CAA codons.

31. An antisense strand as claimed in one of claims 27 to 30.

32. The amino acid sequence corresponding to amino acids 1 to 205 of the amino acid sequence shown in FIG. 2.

33. A process for preparing the cathepsin L as claimed in one of claims 24 to 26 and 32, which comprises culturing ciliates in a suitable medium and subsequently isolating the cathepsin L.

34. The process as claimed in claim 33, wherein the cathepsin L is isolated from the ciliate cells.

35. The process as claimed in claim 33, wherein the cathepsin L is isolated from the medium.

36. The process as claimed in one of claims 33 to 35, wherein the ciliate is Paramecium.

37. The process as claimed in claim 36, wherein the ciliate is Paramecium tetraurelia.

38. The process as claimed in one of claims 33 to 37, wherein the cathepsin L possesses the amino acid sequence as claimed in claim 32.

39. A cathepsin L as claimed in one of claims 24 to 26 and 32 for use as a pharmaceutical.

40. The use of the cathepsin L as claimed in one of claims 24 to 26 and 32 for preparing a pharmaceutical for treating wounds.

41. A wound ointment which comprises cathepsin L as claimed in one of claims 24 to 26 and 32.

42. The use of cathepsin L as claimed in one of claims 24 to 26 and 32 for identifying a cathepsin L inhibitor.

43. A cathepsin L propeptide as claimed in claim 18 or 23 for use as a pharmaceutical.

44. The use of the cathepsin L propeptide as claimed in claim 18 or 23 for preparing a pharmaceutical for treating inflammatory diseases.

45. The use of the cathepsin L propeptide as claimed in claim 18 or 23 for preparing a pharmaceutical for treating metastasizing tumors.

46. The use of the cathepsin L propeptide as claimed in claim 18 or 23 for preparing a pharmaceutical for treating bacterial infections.

47. The use of the cathepsin L propeptide as claimed in claim 18 or 23 for preparing a pharmaceutical for treating infections with parasitic protozoa.

48. The use of the cathepsin L propeptide as claimed in claim 18 or 23 for preparing a pharmaceutical for treating osteoporosis.

49. A process for preparing cathepsin L as claimed in one of claims 24 to 26, which comprises expressing the cathepsin L in a heterologous expression system using a DNA sequence as claimed in claim 29.

50. A process for preparing the cathepsin L propeptide as claimed in one of claim 18 or 23, which comprises expressing the propeptide in a heterologous expression system using a DNA sequence as claimed in claim 20.

51. The process as claimed in claim 50, wherein the expression system is E. coli.

52. The process as claimed in claim 51, wherein a DNA sequence as claimed in claim 21 is introduced into E. coli using a heat-inducible vector.

53. The use of the presequence as claimed in claim 8 for translating into a leader sequence as claimed in claim 9, which leader sequence serves as the signal for secreting recombinantly expressed peptides or proteins in ciliates.

54. The use of the presequence as claimed in claim 10 or 12 for translating into a leader sequence as claimed in claim 15, which leader sequence serves as the signal for secreting recombinantly expressed peptides or proteins in ciliates.

55. The use of the cathepsin L propeptide as claimed in claim 18 or 23, or of parts or analogs thereof, for preparing cathepsin L inhibitors.