1 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical University, No. 1 Tiantanxili, Beijing 100050, China
2 Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
Correspondence
Jozef Anné
jozef.anne{at}rega.kuleuven.be
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ABSTRACT |
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INTRODUCTION |
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The proteasome represents the major non-lysosomal proteolytic system in eukaryotes (reviewed by Hochstrasser, 1995; Voges et al., 1999
). The 20S proteasome forms the catalytic core of the ATP-dependent 26S proteasome that mediates ubiquitin-dependent protein turnover. The proteolytic activity is confined to an inner compartment that is accessible only to unfolded proteins. The counterpart of the proteasome is ubiquitous in Archaea (Dahlmann et al., 1989
), but ubiquitin genes were not found in bacterial genomes sequenced so far. It was therefore unexpected that 20S proteasome homologues were discovered within the genome of Actinomycetes (Nagy et al., 1998
), a lineage of high-GC Gram-positive bacteria, including Streptomyces coelicolor A3(2) (Nagy et al., 1998
, 2003
; De Mot et al., 1999
), Mycobacterium leprae and Mycobacterium smegmatis (Knipfer & Shrader, 1997
), Mycobacterium tuberculosis (Darwin et al., 2003
) and Rhodococcus erythropolis (Zuhl et al., 1997
). No known function could be attributed to the 20S proteasome in bacteria so far, except for the M. tuberculosis proteasome that has been found to protect the organism against oxidative or nitrosative stress (Darwin et al., 2003
). In bacteria, intracellular proteolysis is carried out by at least four other ATP-dependent, compartmentalized proteases of the ClpAP/XP, HslUV, FtsH and Lon families. For example, ClpP in Escherichia coli has been reported to be involved in response to conditions when foreign proteins are overexpressed (Gill et al., 2000
). TepA of B. subtilis, showing sequence similarity to protease ClpP, was reported to be required for efficient translocation and processing of secretory proteins (Bolhuis et al., 1999a
).
Until now the cellular targets for the bacterial 20S proteasome remain to be identified. In this paper, we report on the inactivation of the 20S proteasome in S. lividans TK24 and its role in the integrity of some secretory heterologous proteins including soluble human tumour necrosis factor receptor II (shuTNFRII) and sCT. Moreover, we also investigated whether a difference in susceptibility to proteasome related hydrolysis might exist for proteins secreted either through the Sec- (for shuTNFRII and shuTNFRI) or Tat-dependent pathway (for cCT). We also discuss the potential of a proteasome-deficient mutant to increase the secretory production of relevant heterologous proteins in S. lividans.
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METHODS |
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Plasmid constructions.
The plasmids used and constructed in this study are listed in Table 1.
To inactivate the 20S proteasome of S. lividans, a suicide vector for streptomycetes, pUTPK, was constructed using the cloned prcBA operon, encoding - and
-subunits of the 20S proteasome (Fig. 1
a) (GenBank accession no. AF086832) of S. coelicolor A3(2), which is closely related to S. lividans (Kawamoto & Ochi, 1998
). The prcBA operon was deleted by replacement of 67 % of prcB and 83 % of prcA with the aph (aminoglycoside phosphotransferase) gene from Tn5 (prcBA : : aph allele, Fig. 1a
) conferring resistance to kanamycin. For this purpose, the following steps were carried out. (i) The KpnI fragment of S. coelicolor prcBA operon (5·1 kb) present on pHSS19-prcBA was cloned into the pUC19 KpnI site, resulting in plasmid pUC19-prcBA. (ii) The internal 1261 bp ScaIMluI fragment, containing part of the prcB and prcA ORFs, was removed by partial digestion with ScaI (a site located in pUC19). (iii) Subsequently, it was digested with MluI and the protruding ends were filled in using the Klenow polymerase reaction. (iv) The aph gene fragment present in pFD666 (Denis & Brzezinski, 1992
) was amplified by PCR using oligonucleotides KAN1 (5'-CAGGGGGGCGGAGCCTATGG-3') and KAN2 (5'-TACTGCGGCCGCGATCCAAGC-3'). (v) The amplified fragment was cloned into pGEM-T Easy, resulting in the plasmid pGEM-aph. (vi) After digestion with EcoRI the obtained aph fragment (1227 bp) was purified and blunt-ended prior to ligation to the restricted pUC19-prcBA, giving rise to plasmid pUPK2. (vii) Finally, the prcBA : : aph fragment from pUPK2 was isolated and cloned into the KpnI site of pUC18-tsr, resulting in the suicide plasmid pUTPK. pUTPK also contains a thiostrepton resistance gene encoding 23S A1067 rRNA methylase from Streptomyces azureus (Kieser et al., 2000
).
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For the secretory production of shuTNFRII in S. lividans, the expression vector pCBS2TNFRII was made. To this purpose, the DNA fragment encoding shuTNFRII was generated by PCR using the primers TNFRII-A (5'-GCATGCATTGCCCGCCCAGGTGGCATT-3'; the underlined sequence indicates the restriction enzyme recognition sequence for NsiI) and TNFRII-B1 (5'-AGAAGCTTTCAGCCAGTGCTCCCTTCAGCTGG-3'; the underlined sequence indicates the restriction enzyme recognition sequence for HindIII). The bold characters indicate the TGA stop codon in the complemented reversed sequence) and cloned into pCR2.1 by TA cloning (Invitrogen). The shuTNFRII coding sequence fragment was excised from this plasmid by NsiI. After blunt-ending using T4 DNA polymerase and digestion with HindIII, this fragment was cloned into the DraII-digested, Klenow polymerase blunt-ended and HindIII-digested pBS-CBSS (Lammertyn et al., 1997). As such the shuTNFRII coding sequence could be inserted after the regulatory sequences of Streptomyces venezuelae subtilisin inhibitor (vsi) gene consisting of the vsi promoters, ribosome-binding site, signal sequence and two codons of the mature vsi gene. The in-frame fusion was confirmed by DNA sequence analysis using the primer DraII SEQ (5'-Cy5-GATGGCACTCACTCTCCGCACC-3'). Next, the entire expression cassette excised by HindIII and BamHI digestion was cloned into pIJ486 (Ward et al., 1986
), similarly digested, resulting in plasmid pCBS2TNFRII.
Similarly, the expression vector pCBS2TNFRI was constructed for secretory production of soluble human tumour necrosis factor receptor I (shuTNFRI). The expression plasmid pMSA was constructed as described previously (Hong et al., 2003) for the secretion of salmon calcitonin (sCT).
Growth analysis.
To evaluate the effect of the 20S proteasome on the growth of S. lividans, the wild-type strain TK24, strain PRO41 with a deleted 20S proteasome and strain PRO41R, containing an intact 20S proteasome on a plasmid for complementation of the chromosomally deleted 20S proteasome, were grown in casein medium and NM medium. At indicated time intervals during growth, cells of 5 ml cultures were collected by centrifugation, washed twice with 50 mM Tris/HCl, pH 7·0, and then dried at 100 °C to a constant weight.
Growth was also assessed qualitatively on solid media. A 20 µl volume of spore suspension was spread on the surface of solid minimal and R2YE medium (Kieser et al., 2000), and plates were incubated at 28 and 37 °C for 7 days. The conditions used were essentially those described by Knipfer & Shrader (1997)
. To analyse the effect of growth under stress conditions, the R2YE medium was supplemented with ethanol (7 %, v/v), cadmium chloride (0·1 mM), puromycin (0·1 or 0·01 mg ml1) or sodium chloride (6 or 7 %, w/v) for the induction of stress.
Proteasome activity assays.
The 20S proteasome activity was detected by zymography using Suc-LLVY-AMC (succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin) as a substrate, as described previously by Pouch et al. (2000). The wild-type strain TK24, deletion mutant PRO41 and the complemented strain PRO41R were cultured for 72 h in NM medium. The mycelium was subsequently collected by centrifugation, washed twice, and resuspended in 5 ml 50 mM Tris/HCl (pH 8·0). The cells were lysed on ice in a sonicator (B. Braun Biotech International Gmbh) and the cell debris was removed by centrifugation at 4 °C. Cell extracts were analysed by native 8 % PAGE and a subsequent overlay of the gel with 5 ml 50 mM Tris/HCl (pH 8·0) supplemented with 0·1 mM fluorogenic substrate Suc-LLVY-AMC. After incubation for 30 min at 37 °C, the result of the proteasome activity was visualized under UV illumination.
Western blot analysis.
To assess the secretory production of shuTNFRII and shuTNFRI in the wild-type and proteasome mutants of S. lividans TK24, Western blot analysis was performed. At the indicated time intervals, the culture supernatants of S. lividans containing the specific vector were sampled and immediately stored at 20 °C. To 1 ml of ice-chilled supernatants 0·5 ml ice-cold 60 % TCA was added and subsequently thoroughly mixed. After an additional hour on ice, the mixture was centrifuged at 4 °C for 10 min at 12 000 g. The pellets were washed in 95 % ethanol, dried in vacuum and dissolved in 2x loading buffer. Proteins were separated by 10 % SDS-PAGE (Laemmli, 1970) and then blotted onto nitrocellulose membranes using a semi-dry electroblotter (Bio-Rad Laboratories). Efficient transfer of the proteins to the membrane was inspected by non-permanent staining with 0·5 % Ponceau S in 1 % acetic acid. The immunoreactive proteins were detected using diluted solution of goat anti-huTNFRII and huTNFRI antibodies (R&D Systems) followed by alkaline phosphatase-conjugated rabbit anti-goat antibodies (Sigma).
Enzyme immunoassay (EIA).
Recombinant sCT secreted by wild-type and proteasome mutants of S. lividans TK24 was quantified by EIA. At the indicated time intervals, the culture supernatants of S. lividans strains containing sCT secretory vector pMSA were sampled and immediately stored at 20 °C. Supernatants were diluted in the assay buffer supplied by the manufacturer and detected with the peptide enzyme immunoassay kit (Peninsula Laboratories) as described by the manufacturer.
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RESULTS |
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Phenotypic characterization of the S. lividans proteasome mutants
To confirm that disruption of the prcBA operon inactivated the 20S proteasome, the proteasomal activity of wild-type and mutant strains was characterized by zymography. Cell lysates prepared from S. lividans TK24, the null mutant (PRO41) and the complemented strain (PRO41R) were compared for their ability to release the fluorophore AMC from the proteasome-specific substrate Suc-LLVY-AMC under the action of the chymotrypsin-like activity of the proteasome (Tamura et al., 1995). The free AMC fluorescence can efficiently be detected at 380 nm excitation and a 460 nm emission filter.
The release of the AMC moiety was detected by zymography and the results (Fig. 3) indicated that replacement of the prcBA operon with the aph gene in PRO41 abolished the 20S proteasomal activity of S. lividans TK24. On the other hand, in PRO41R, an S. lividans strain that contained the plasmid pPROAPR carrying prcBA operon of S. coelicolor A3(2), the proteosome activity was restored. Moreover, the proteasomal activity in PRO41R was higher than in S. lividans TK24, probably due to the reintroduction of the prcBA operon on a high copy number plasmid derived from pFD666.
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The influence of the 20S proteasome on the yield of secreted shuTNFRII
The heterologous expression cassette of shuTNFRII was constructed by fusion of the coding sequence of the extracellular domain of human TNFRII downstream of the signal peptide and the first 2 aa of the mature S. venezuelae subtilisin inhibitor (Vsi) (Van Mellaert et al., 1998). The expression plasmid pCBS2TNFRII constructed from pIJ486, containing a pIJ101 ori, was introduced by transformation into S. lividans TK24, PRO41 and PRO41R. The three strains carrying pCBS2TNFRII multiplied at similar growth rates in casein medium, resembling the growth curve of their parent strains (data not shown). The amount of secreted shuTNFRII was detected by Western blot analysis, and the results (Fig. 4
) showed that the three strains differed markedly. In S. lividans TK24, the secretory production of shuTNFRII was almost undetectable, suggesting some bottlenecks in the expression and/or secretion pathway. When the proteasome-deficient strain PRO41 was used to express shuTNFRII in casein medium, a band with a predicted molecular mass of shuTNFRII (ca. 26 kDa) could be detected by Western blot analysis. A full-length product, but also a specific smaller band (ca. 22 kDa), could be detected through 2472 h. The latter band was probably a breakdown product of the intact shuTNFRII. When the proteasome operon was reintroduced into the deficient strain resulting in S. lividans PRO41R, shuTNFRII was again undetectable. This result indicates that 20S proteasome hampers the extracellular accumulation of shuTNFRII in S. lividans TK24.
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The influence of proteasome on the yield of secreted sCT
For secretory production of C-terminal amidated sCT, the promoter, ribosome-binding site and signal peptide of melC1 from Streptomyces antibioticus (Bernan et al., 1985) was used to construct the expression cassettes of sCT-Gly and rat
-amidating enzyme (
-AE). The latter enzyme is responsible for the conversion of glycine-extended substrates to the biologically active, C-terminal amidated products. The expression plasmid pMSA (Hong et al., 2003
), which contains both expression cassettes, was constructed from the pIJ101-ori containing pIJ680. S. lividans TK24, strain PRO41 and PRO41R were each transformed with pMSA, and the three strains carrying pMSA grew at similar growth rates in NM and casein medium, and resembled the growth curve of their parent strains (data not shown). sCT yield was detected by EIA. The results (Fig. 5
) showed that, in both NM and casein medium the sCT expression level by the proteasome-deficient strain PRO41 was much higher than that of the wild-type TK24 strain or the complemented proteasome-deficient strain PRO41R. This result indicates that the production of sCT in S. lividans TK24 is also hampered by the 20S proteasome.
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DISCUSSION |
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Streptomyces has a naturally high secretion capacity and as a consequence is used as an efficient host for the production of some heterologous proteins (Binnie et al., 1997; Pozidis et al., 2001
; Van Mellaert & Anné, 2001
). Most proteins are secreted via the Sec-dependent pathway (reviewed by Mori & Ito, 2001
), but also the recently detected Tat pathway is functional in Streptomyces (Schaerlaekens et al., 2001
). The latter pathway can translocate folded proteins, while the Sec-dependent pathway translocates newly synthesized polypeptides in an unfolded conformation with the help of chaperones. So far, heterologous protein secretion has mostly been investigated using signal peptides that direct the secretion to the Sec pathway, but the Tat pathway can also translocate heterologous proteins, although less efficiently for the proteins tested (Schaerlaekens et al., 2004
).
To investigate the possible influence of the proteasome on heterologous protein production, the yields of shuTNFRII, sCT and shuTNFRI were compared with the wild-type S. lividans TK24 strain, a 20S proteasome null mutant obtained by replacement of the 20S proteasome with the aph gene, and with a complemented 20S proteasome deletion strain carrying the 20S proteasome gene on a multicopy plasmid.
Two different types of signal peptide-dependent secretion have been used in the experiments described. The Vsi signal peptide of the S. venezuelae subtilisin inhibitor, which is Sec-dependent, has been successfully used to produce mouse TNF- in large amounts (Lammertyn et al., 1997
). However, when the same construct strategy (fusion of the coding sequence of heterologous proteins after the signal peptide and 2 aa of mature protein of Vsi) was used to express some other eukaryotic proteins including huTNF-
, huTNF-
, shuTNFRI and shuTNFRII, the yield was variable (Lammertyn, 2000
). This indicates the existence of bottlenecks in the expression and/or the secretion of a number of heterologous proteins in S. lividans.
sCT was secreted under the control of the melC1 promoter and the Tat-dependent signal peptide of MelC1. The latter is essential for the secretion of the S. antibioticus tyrosinase MelC2 by forming a MelC1MelC2 complex before export (Schaerlaekens et al., 2001). MelC1 has been successfully used to secrete some heterologous proteins in S. lividans (Chang & Chang, 1988
; Hong et al., 2003
; Schaerlaekens et al., 2004
).
The yield of shuTNFRII using expression/secretion signals of Vsi and of sCT directed by MelC1 differed significantly for S. lividans TK24, the proteasome-deficient strain PRO41 and the 20S proteasome complemented strain PRO41R, with increased levels of heterologous proteins observed only in the 20S proteasome-deficient strain, but not in the wild-type nor in the complemented strain. This difference suggests that the 20S proteasome in S. lividans may be important for the production of some heterologous proteins. The reason for this might be that the heterologous proteins may be captured by the 20S proteasome and degraded before being recognized by the secretion machinery of S. lividans, including the Sec-dependent as well as the Tat-dependent pathway.
However, the production of another heterologous protein tested, i.e. shuTNFRI, was not affected by the presence of the 20S proteasome. Hence, the 20S proteasome seems to be a secretion bottleneck for some heterologous proteins while not for others, probably depending on the characteristic of the protein to be secreted. This protease vulnerability and the presence of secretion bottlenecks is consistent with what has been reported for B. subtilis (Bolhuis et al., 1999b), in which the secretion bottlenecks differed for each heterologous protein tested and the identified secretion bottlenecks were at late stages in the secretion process, such as efficient processing by signal peptidase, passage through the cell wall, proteases secreted into the medium as well as cell-associated proteases, which are responsible for the degradation of secreted heterologous proteins. In S. lividans, similar secretory bottlenecks may exist when heterologous proteins are expressed, although our knowledge about this is quite fragmentary. The endogenous proteases produced by S. lividans intracellularly (Butler et al., 1994
), extracellularly (Lichenstein et al., 1992
; Butler et al., 1995
; Krieger et al., 1994
; Mori & Ito, 2001
) or cell wall-associated (Binnie et al., 1995
) have been investigated to be bottlenecks for the production of some heterologous proteins. The results obtained here show that the 20S proteasome seems to be a bottleneck for some heterologous proteins produced in S. lividans such as shuTNFRII and sCT but not for others as illustrated with shuTNFRI. For the proteins vulnerable for proteasome degradation, the expressed pre-protein may be malfolded prior to or during secretion, and not efficiently adapted to the Sec- or Tat-dependent pathway apparatus, and as a consequence subjected to the degradation of 20S proteasome.
In conclusion, this report clearly indicates that the proteasome could be an as yet not identified bottleneck in the expression and secretion of some heterologous proteins by S. lividans. More importantly, the results presented clearly point out that the optimization of the production of specific heterologous protein(s) is likely to require engineered S. lividans strains, in which the proteasome has been removed. With the genetic amenability of this organism and increasing knowledge of the bottlenecks in expression and secretion, it will be possible to engineer the dedicated S. lividans host strains for the production of a wide range of heterologous proteins at commercially significant levels.
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ACKNOWLEDGEMENTS |
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Received 15 March 2005;
revised 27 May 2005;
accepted 13 June 2005.
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