Gene encoding chondroitinase ABC and uses therefor

Abstract

nucleic acid sequences coding for the chondroitinase ABC gene and isolated chondroitinase ABE protein produced in a host cell transformed with a nucleic acid vector directing the expression of a nucleotide sequence coding for chondroitinase ABE protein described. Chondroitinase ABC prepared by chemical synthesis also described. Monoclonal and polyclonal antibodies which are specifically reactive with chondroitinase ABC protein are disclosed. The isolated chondroitinase ABC can be used in methods of treating intervertebral disc replacement, promoting neurite regeneration, and detecting galactosaminoglycans.

Description

This application is a continuation of application Ser. No. 08/184,435 filed on Jan. 14, 1994 now abandoned Entitled: Gene Encoding Chondroitinase ABC And Uses Therefor, which is a divisional of Ser. No. 08/074,349 filed Jun. 8, 1993 now abandoned.

BACKGROUND OF THE INVENTION

Chondroitin lyase (EC 4.2.2.4) or chondroitinase ABC is an enzyme which catalyzes the depolymerization of chondroitin sulfate. Through β-elimination of 1,4 hexosaminidic bonds, chondroitinase ABC degrades chondroitin, chondroitin 4-sulfate (chondroitin A sulfate), dermatan sulfate (chondroitin B sulfate), chondroitin 6-sulfate (chondroitin C sulfate) and hyaluronate to the respective unsaturated disaccharides (Δdi-OS for chondroitin, Δdi-4S for chondroitin A sulfate, Δdi-4-6S for chondroitin B sulfate and Δdi-6S for chondroitin C sulfate, respectively). The enzyme has been isolated in various strains of bacteria (Neuberg, C. et al., (1914) Biochem. Z. 67: 82-89) (Neuberg, C. et al. (1931) Biochem, Z. 234: 345-346; Yamagata, T. et al., (1968) J. Biol. Chem. 243: 1523-1535) including Proteus vulgaris (Yamagata, T. et al. (1968) J. Biol. Chem. 243: 1523-1535; Thurston, C. F. (1974) J. Gen. Microbiol. 80: 515-522; Sato N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059; Sato N. et al. (1986) Biotechnol. Bioeng. 28: 1707-1712; Sato, N. et al. (1986) J. Ferment. Technol. 64: 155-159).

Chondroitin sulfate consists of alternating β 1-3 glucuronidic and β 1-4 N-acetylgalactosaminidic bonds, and is sulfated at either C-4 or C-6 of the N-acetylgalactosamine pyranose. Chondroitin sulfate is known to be widely distributed in mammalian tissue, such as in skin, cornea, bone and especially in cartilage. Thus, chondroitinase ABC has been used as an experimental reagent for the determination or quantitation of total amount of galactosaminoglycans in the field of orthopedic surgery (Linker, A. et al. (1960) J. Biol. Chem. 235: 3061-3065; Saito, H. et al. (1968) J. Biol. Chem. 243: 1536-1542; Pettipher, E. R. et al. (1989) Arthritis Rheum. 32: 601-607; Caterson, B. et al. (1990) J. Cell Science 97: 411-417; and Seibel, M. J. et al. (1992) Arch. Biochem. Biophys. 296: 410-418).

Recently, chondroitinase ABC has been reported to be a potential reagent for chemonucleolysis, an established treatment for intervertebral disc displacement (Kato, F. et al. (1990) Clin. Orthop. 253: 301-308; Henderson, N. et al. (1991) Spine 16: 203-209). However, for the utilization of chondroitinase ABC as a clinical reagent, there are many problems to be overcome. For example, the preparation of chondroitinase ABC from P. vulgaris requires tedious and intricate procedures, since the cellular content of the enzyme is low. Therefore, an efficient method for the efficient preparation of highly purified chondroitinase ABC is now sought.

SUMMARY OF THE INVENTION

This invention pertains to nucleic acid sequences coding for the chondroitinase ABC gene and isolated chondroitinase ABC protein produced in a host cell transformed with a nucleic acid vector directing the expression of a nucleotide sequence coding for chondroitinase ABC. Chondroitinase ABC prepared by chemical synthesis is also provided. This invention further provides monoclonal and polyclonal antibodies which are specifically reactive with chondroitinase ABC. The isolated chondroitinase ABC can be used in methods of treating intervertebral disc displacement and promoting neurite regeneration or in method of detecting the presence of galactosaminoglycans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-A and 1-B show the primers used for polymerase chain reaction (PCR) amplification of chondroitinase ABC from P. vulgaris genomic DNA5 3′-GACTACGTCAGGCTTTT AAAT-3 5′)(SEQ ID NO:11) (FIG. 1-B) and 1 μg of P. vulgaris genomic DNA as a template, PCR analysis was performed and PCR products were analyzed by agarose gel electrophoresis. No non-specific PCR products were observed.

We then diluted γEMBL3 recombinant phage stock library. The diluted library was used for PCR screening. An unique 54 bp fragment was clearly detected until the dilution of 1/10³(2×10⁵ pfu) phage stock solution as a template. The diluted phage solution was divided by 1/10(2×10⁴ pfu) and was infected into E. coli P2392. They were then subjected to plaque hybridization using ³²P-labeled probe (5′-CATTTGATCCTAAAAATCTGATGCA-3′)(SEQ ID NO:9)(FIG. 1-B). The recombinant phages were chosen at random and analyzed by restriction mapping and Southern blotting. All phages contained common 4.2 kb EcoRV-EcoRI, 1.1 kb ClaI, and 2.0 kb EcoRV-HindIII fragments which hybridized strongly with the probe(SEQ ID NO:9). The restriction maps of three types of SalI fragments are shown in FIG. 1-C. Southern hybridization patterns of restricted genomic DNA from P. vulgaris matched the restriction map of these fragments. This result suggests that the 4.2 kb EcoRV-EcoRI fragment originated in the P. vulgaris genome, and therefore, the chondroitinase ABC gene exists as a single copy. When purified chondroitinase ABC from P. vulgaris was analyzed by SDS-PAGE, two types of chondroitinase ABC protein, one 100 kd protein and one subunit-like protein at 80 kd and 20 kd, were observed. The amino acid composition of the 100 kd protein and the subunit-like protein (80 kd and 20 kd) were quite similar, and the N-terminal amino acid sequences of the 100 kd and 20 kd proteins were identical. The results indicate that the two forms of chondroitinase ABC were not derived from two separate chondroitinase ABC genes.

The 5.2 kb SalI-EcoRI fragment in the recombinant γEMBL3 (No. 11-5) (FIG. 1-C) was subcloned into pSTV29 for sequencing and the resulting hybrid plasmid was designated pCHS6. The entire 3,063 bp nucleotide sequence of the coding region for the chondroitinase ABC gene as well as 224 and 200 nucleotides of the upstream and downstream regions, respectively, and the deduced amino acid sequence of chondroitinase ABC are shown in FIG. 7 (SEQ ID NO:1). The 25-mer oligonucleotide probe (SEQ ID NO:9) hybridized to nucleotide 314-337. The 16/18 nucleotide of primer A and the 17/18 nucleotide of primer B were the same in nucleotides 297-313 and 333-349. The G+C content of the chondroitinase ABC gene was 38.6%. The open reading frame encoded a polypeptide with a molecular weight of 115,218, which represents a precursor polypeptide containing a signal peptide sequence that is subsequently cleaved off at Ala²⁴-Ala²⁵ during secretion of the mature chondroitinase ABC protein having a molecular weight of 112,365.

EXAMPLE 2

Analysis of the transcription region of the chondroitinase ABC gene In order to confirm the potential promoter region of the chondroitinase ABC gene, we amplified the region of nucleotide 112-283 using PCR. The PCR product was blunt-ended with T4 DNA polymerase and inserted into the SmaI site of the promoter selection vector, pMC 1871, and the hybrid plasmid, designated pCHSP, was introduced into E. coli JM109 (FIG. 2)(SEQ ID NO:14). The transformant was then cultured in an LB medium containing 25 μg/ml tetracycline at 37° C. for 14 hr, and β-galactosidase activity was assayed (Table I). Although the β-galactosidase activity of the E. coli transformant carrying pMC1871 was not detectable, the E. coli transformant carrying pCHSP produced β-galactosidase. This result indicates that the chondroitinase ABC gene can function as a promoter in E. coli cells. However, there is a possibility that the promoter recognized in E. coli cells may not be the promoter in P. vulgaris. To confirm that the promoter is recognized in P. vulgaris, primer extension analysis was carried out (FIG. 3) (SEQ ID NO:15). The transcription start point was localized to an adenosine 41 bp upstream from the start codon, ATG (FIG. 4) (SEQ ID NO:16). The potential pribnow box (TTTAAT) (nucleotides 169-174) was located 12 bp upstream from the transcription start point (FIG. 4) (SEQ ID NO: 16). However, the −35 consensus sequence was not found near 35 bp upstream of the start point except for 47 bp upstream of the start point (TAGGCA) (FIG. 4) (SEQ ID NO:16). The Shine-Dalgarno ribosomal binding site (AGGAGA) (nucleotides 213-218) was found 9 bp upstream from the initiation codon, ATG (FIG. 4) (SEQ ID NO: 16). A terminator-like palindrome sequence consisting of an 11 nucleotide stem with a 4 nucleotide loop structure (stacking energy 24 kcal/mol) was located 9 nucleotides downstream from the stop codon, TGA (FIG. 4) (SEQ ID NO:16). Judging from the secondary structure prediction, this stem-loop structure resembles a σ-dependent transcription terminator.

TABLE I
β-Galactosidase productivity of E-coli transformants
		β-Galactosidase activity
	Strain	Activity	Specific activity/
	E. coli JM109	(U/mi-culture)	(U/mg-protein)
	/pMC1871	0	0
	/pCHSP	0.2	0.4
	1 U is defined as the amount that produced 1 μmol of α-nitrophenol per h.

EXAMPLE 3

Production of chondroitinase ABC from E. coli transformant To demonstrate that the isolated gene codes for chondroitinase ABC, we constructed pCHSΔ6 and pCHS26 (FIG. 5). pCHSΔ6 was constructed by removing the SalI-EcoRV region (about 1 kb) upstream from the promoter region from the chondroitinase ABC gene. While pCHS26 was constructed by removing the HindIII-EcoRI region which corresponded to about one third of the 3′-terminal region of the chondroitinase ABC structural gene. These plasmids (pCHS6, pCHSΔ6 and pCHS26) were introduced into E. coli XL 1-Blue, and E. coli transformants were cultured in chondroitin or glucose medium, and chondroitinase ABC activities were assayed using the crude extract. The culture fluids of the chondroitin medium were also analyzed to determine degradation products of chondroitin 6-sulfate (Table II). The E. coli transformant carrying pCHS6 (containing a 1.0 kb fragment upstream from the promoter) produced the chondroitinase ABC when cultured in chondroitin medium, however, no chondroitinase ABC activity was observed when the transformant was cultured in glucose medium. In contrast, the E. coli transformant carrying pCHSΔ6 produced chondroitinase ABC when cultured in either chondroitin or glucose media. The production levels of chondroitinase ABC, cultured in chondroitin media, were 2.6 fold(/pCHS6) and 187 fold(/pCHSΔ6) higher than that of P. vulgaris. Even cultured in glucose medium, the production level of chondroitinase ABC in the E. coli transformant carrying pCHSΔ6 was 187 fold higher than that of P. vulgaris cultured in chondroitin medium. This result suggests that the regulatory sequence might be in the SalI-EcoRV region. Although chondroitin 6-sulfate added to the medium was degraded (p/CHS6 and /pCHSΔ6), E. coli transformants were not able to utilize chondroitin sulfate as a carbon source.

TABLE II
Chondroitinase ABC Activity of E. coli Transformants
	Intracellular chondroitinase ABC activity
	Chondroitin medium	Glucose	Cultured medium
	(0.3%)	medium (0.3%)	Amount of 4,5Δ
		Specificb		Specific	chondroitin-6
Strain	Activitya	activity	Actiity	activity	(μg/ml-culture)
E. coli	0	0	0	0	0
XL1-Blue
/pSTV29	0	0	0	0	0
/pCHS6	4.1 × 10−3	1.6 × 10−2	0	0	192.7
/pCHS26	0	0	0	0	0
/pCHSΔ6	0.3	1.2	0.3	0.5	1542.4
P. vulgaris	1.6 × 10−3	1.2 × 10−2	0	0	1738.4
a1 U: enzyme activity producing 1 μmol, 4,5Δ chondroitin-6 per min
bU/mg-protein

It has been reported that the Bacteriodes thetaiotaomicron chondroitin lyase II gene is adjacent to the chondrosulfatase gene which may be a part of an operon (Guthrie, E. P. et al. (1987) J. Bacteriol. 169: 1192-1199). These same investigators reported that the promoter for this gene recognized in E. coli may not be the promoter from which the chondroitin lyase II gene is transcribed from in B. thetaiotaomicron (Ld.) In fact, a putative open reading frame 12 bp upstream from the initiation codon, ATG, was found in the chondroitinase ABC gene (FIG. 4) (SEQ ID NO: 16). However, primer extension analysis revealed that the transcription start point is located 41 bp upstream from the initiation codon in P. vulgaris(FIG. 3) (SEQ ID NO: 15). Even though the chondroitinase ABC gene from P. vulgaris cells was also part of an operon, chondroitinase ABC gene was transcribed 41 bp upstream from the initiation codon in P. vulgaris cells.

The secondary structure of chondroitinase ABC was estimated by the method of Chou and Fasman (Annu. Rev. Biochem. 47: 251-276 (1978)). A highly complex region was found between amino acid residues 450 and 850. The pCHS26 lacks one-third of the chondroitinase ABC gene encoding the C-terminal region (amino acid residues 646-1021). Removing this region of the enzyme caused the disappearance of chondroitinase ABC activity (Table II). This result suggests that there might be an active site in this region.

Recombinant chondroitinase ABC produced by E. coli carrying pCHSΔ6 was analyzed by SDS-PAGE followed by immunoblotting (FIG. 6). The immunoblotting patterns of recombinant and native chondroitinase ABC (100 kd) were quite similar. Our previous report showed chondroitinase ABC purified from P. vulgaris was a subunit structure consisting of a 90 kd and a 20 kd protein by SDS-PAGE (Sato, N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059), because this subunit protein would not be separated even using gel filtration and other chromatographic techniques. However, by analysis of the N-terminal sequence, we found that the 100 kd protein and the 20 kd protein had the same N-terminal amino acid sequence. By immunoblot analysis, the 80 kd protein also reacts with IgG specific to the 100 kd protein. Furthermore, genomic restriction analysis suggested that chondroitinase ABC gene was a single gene. When we extracted the 100 kd band of chondroitinase ABC from the acrylamide gel and electrophoresed it again in SDS-PAGE, 80 kd and 20 kd bands appeared. The purified chondroitinase ABC contained no protease activity. These results suggest that chondroitinase ABC was partially digested not enzymatically, but physically in the course of sample preparation for SDS-PAGE.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. An isolated nucleic acid fragment encoding chondroitinase ABC, comprising the nucleotide sequence of SEQ ID NO: 1.

2. An expression vector comprising the nucleic acid as defined in claim 1 operably linked to a regulatory sequence.

3. A host cell transformed with the expression vector as defined in claim 2.

4. A host cell of claim 3 23 wherein the cell is eukaryotic.

5. A host cell of claim 3 23 wherein the cell is prokaryotic.

6. A method of producing chondroitinase ABC protein comprising: culturing the host cell as defined in claim 2 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

7. An isolated nucleic acid encoding chondroitinase ABC comprising a nucleotide sequence which differs from the nucleotide sequence of SEQ ID NO: 1, due to degeneracy in the genetic code.

8. An expression vector comprising the nucleic acid as defined in claim 7 operably linked to a regulatory sequence.

9. A host cell transformed with the expression vector as defined in claim 8.

10. A method of producing chondroitinase ABC protein comprising:

culturing the host cell as in defined in claim 9 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

11. An isolated nucleic acid fragment comprising the coding region of chondroitinase ABC and having a nucleotide sequence consisting of nucleotides 297-3288 73 to 3066 of SEQ ID NO: 1.

12. An expression vector comprising the nucleic acid of claim 11 operably linked to a regulatory sequence.

13. A host cell transformed with the expression vector of claim 12.

14. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 13 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

15. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides 297-3288 73 to 3066 of SEQ ID NO: 1, due to degeneracy in the genetic code.

16. An expression vector comprising the nucleic acid as defined in claim 15 operably linked to a regulatory sequence.

17. An isolated nucleic acid fragment comprising nucleotides 2160-3288 of SEQ ID NO: 1.

18. An expression vector comprising the nucleic acid as defined in claim 17 operably linked to a regulatory sequence.

19. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides 2160-3288 of SEQ ID NO: 1, due to degeneracy in the genetic code.

20. An expression vector comprising the nucleic acid as defined in claims operably linked to a regulatory sequence.

21. An isolated nucleic acid fragment comprising the coding region of the nucleotide sequence of SEQ ID NO: 1.

22. An expression vector comprising the nucleic acid as defined in claim 21 operably linked to a regulatory sequence.

23. A host cell transformed with the expression vector as defined in claim 22.

24. A method of producing chondroitinase ABC protein comprising:

culturing the host cell as in defined in claim 23 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

25. An isolated nucleic acid comprising a nucleotide sequence which differs from the coding region of SEQ ID NO: 1, due to degeneracy in the genetic code.

26. An expression vector comprising the nucleic acid as defined in claim 25 operably linked to a regulatory sequence.

27. A host cell transformed with the expression vector as defined in claim 26.

28. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in claim 27 under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.

29. An isolated nucleic acid fragment encoding chondroitinase ABC, wherein the nucleic acid comprises a nucleotide sequence of the insert of pCHS6 obtained from E. coli XL1-Blue/pCHS6 deposited at Accession NO. FERM BP-4170 .