1 Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, via Cinthia, 80126 Napoli, Italy
2 ISPAAM, Consiglio Nazionale delle Ricerche, via Argine 1085, 80147 Napoli, Italy
Correspondence
Paola Giardina
giardina{at}unina.it
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ABSTRACT |
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The EMBL accession numbers for the nucleotide sequences reported in this paper are AJ634913 (posl) and AJ748587 (pcsl).
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INTRODUCTION |
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Pleurotus ostreatus and Phanerochaete chrysosporium are white-rot basidiomycetes, which belong to different subclasses of ligninolytic micro-organisms, producing distinct patterns of ligninolytic enzymes. They produce extracellular proteases, which are believed to be involved in the regulation of the ligninolytic activities of these fungi (Palmieri et al., 2000, Dosoretz et al., 1990
). In particular, it has been demonstrated that different laccase isoenzymes from Pl. ostreatus can be specifically degraded or activated during fungal growth by proteases present in the culture broth (Palmieri et al., 2000
, 2003
). In a recent study, we reported the purification and characterization of the main Pl. ostreatus extracellular protease PoSl (Palmieri et al., 2001
).
On the basis of structural and kinetic properties, PoSl appears to be a serine protease belonging to the subtilase family. This enzyme seems to play a key role in the regulation process of Pl. ostreatus laccase activities. A similar relationship was observed for lignin peroxidases (LiPs) in Ph. chrysosporium: in this case, the extracellular proteases caused an almost complete disappearance of LiP activity due to degradation of all LiP isoenzymes (Dosoretz et al., 1990).
This paper reports evidence concerning the role played by PoSl in the activation of Pl. ostreatus extracellular proteases. The posl gene and cDNA were cloned and sequenced, and mass spectrometric analysis was employed to validate the deduced amino acid sequence and to identify post-translational modifications. Furthermore, analyses by homology search allowed us to define a new subtilase subfamily which includes proteases from ascomycete and basidiomycete fungi.
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METHODS |
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Pl. ostreatus cultures were carried out in the basal medium as previously described (Palmieri et al., 1997), or with the addition of 150 µM copper sulphate or 1 mM vanillic acid (4-hydroxy-3-methoxybenzoic acid). Ph. chrysosporium cultures were carried out in 0·24 % potato glucose broth in the presence of 0·05 % yeast extract.
Protease assay.
Protease activity was assayed using SucAAPFpNA (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; Sigma) or azoalbumin as substrates as follows:
a) The assay mixture contained 5 mM SucAAPFpNA, 10 mM CaCl2 and 50 mM Tris/HCl buffer, pH 8·0, in a final volume of 1 ml. Hydrolysis of the substrate was followed by absorbance increase at 405 nm (405=8800 M1 cm1).
b) A 400 µl volume of 15 mg ml1 azoalbumin (Sigma) in 50 mM MOPS buffer, pH 7·0, was incubated with 250 µl of the enzyme sample at 37 °C for 30 min. To stop the reaction, 650 µl 20 % TCA was added and the undigested precipitated substrate was removed by centrifugation at 10 000 r.p.m. for 15 min. Six hundred and fifty microlitres of supernatant was added to 350 µl 10 M NaOH and the absorbance at 440 nm was measured. The control assay was performed without any enzyme in the reaction mixture and used as reference. One unit of enzyme activity was defined as the amount of enzyme needed to increase the A440 by 0·01.
Zymographic analysis.
Samples underwent electrophoresis in 10 % gelatin-containing polyacrylamide gel at alkaline pH under non-denaturing conditions. The separating and stacking gels contained 12·5 % acrylamide solution in 50 mM Tris/HCl, pH 9·5, and 9 % acrylamide solution in 18 mM Tris/HCl, pH 7·5, respectively. The electrode reservoir solution was 25 mM Tris/190 mM glycine, pH 8·4. After electrophoresis, gels were incubated for 16 h at 37 °C in 50 mM Tris/HCl, pH 7·6, buffer containing 200 mM NaCl and 5 mM CaCl2. Gels were then stained for 30 min with 30 % methanol/10 % acetic acid containing 0·5 % Coomassie Brilliant Blue R-250 and destained in the same solution without dye. Clear bands on the blue background represent areas of gelatinolysis.
Cloning of the posl gene.
Amplification experiments of Pl. ostreatus genomic DNA were performed using the oligonucleotide pair 1, as primers (Table 1). The 800 bp fragment obtained was cloned into the pGEM-T Easy Vector (Promega) and sequenced. This fragment, labelled by the random priming method, was used as probe to screen a Pl. ostreatus genomic library (Giardina et al., 1995
). A further screening of the genomic library was performed using, as probe, a 700 bp fragment obtained by KpnI digestion of the amplified cDNA. Colony hybridization experiments were carried out in 5x SSC at 65 °C (where 1x SSC is 0·15 M NaCl, 0·015 M sodium citrate).
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First-strand cDNA synthesized from Pl. ostreatus total RNA using the gene-specific primer posl 1D down (Table 1) was used to perform rapid amplification of 5' cDNA end (5'-RACE). Terminal transferase (Roche) was used to add a homopolymeric A-tail to the 3' end of the cDNA. Tailed cDNA was then amplified by PCR, using oligonucleotide pair 7 (Table 1
), and the resulting product was reamplified using oligonucleotide pair 8 (Table 1
), cloned in pGEM-T Easy Vector and sequenced.
DNA preparation, subcloning and restriction analyses were performed by standard methods according to Sambrook et al. (1989). Sequencing by the dideoxy chain-termination method was performed by the CEINGE Sequencing Service (Naples, Italy) using universal and specific oligonucleotide primers.
Amino acid sequence analysis.
Automated N-terminal degradation of the protein was performed using a Perkin-Elmer Applied Biosystem 477A pulsed liquid protein sequencer equipped with a model 120A phenylthiohydantoin analyser for the on-line identification and quantification of phenylthiohydantoin (PTH) amino acids.
Matrix-assisted laser desorption ionizationmass spectrometry (MALDIMS) analysis.
MALDIMS analyses were carried out with a Voyager DE MALDI Time of Flight mass spectrometer (PerSeptive Biosystems) on both the protein and the peptide mixtures obtained from proteolytic hydrolyses.
Molecular mass determination of whole protease was performed by loading a mixture of sample solution and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) on a sample slide and drying the slide in vacuo. Mass range was calibrated using apomyoglobin from horse heart (mean molecular mass, 16 952·5 Da) and human serum albumin (mean molecular mass, 66 431·0 Da).
Peptide mixtures derived from each hydrolysis were lyophylized and successively dissolved in 0·2 % trifluoroacetic acid, and 1 µl sample solution was mixed with 1 µl 10 mg ml1 -cyano-4-hydroxy-cinnamic acid (CHCA) solution on a sample slide and left to dry under vacuum. The CHCA solution was prepared in acetonitrile : 0·1 % TFA, 70 : 30 (v/v). Mass spectra were acquired in a linear mode, and calibrated from 379·35 and 5734·59, the m/z values of the CHCA dimer and bovine insulin, respectively.
Enzymic hydrolysis.
A PoSl sample (1 nmol) was reduced with 100 mM DTT at 37 °C for 2 h under a nitrogen atmosphere in 0·25 M Tris/HCl (pH 8·5), 1·25 mM EDTA, containing 6 M guanidinium chloride, and then alkylated for 30 min by using an excess of iodoacetamide at room temperature in the dark. The protein sample was desalted by loading the reaction mixture onto a PD-10 prepacked column (Pharmacia), equilibrated and eluted in 0·4 % ammonium bicarbonate, pH 8·5.
Enzymic digestions with trypsin and endoproteinase Glu-C were carried out in 0·4 % ammonium bicarbonate, pH 8·5, at 37 °C overnight using an enzyme/substrate molar ratio of 1 : 50.
The tryptic mixture was deglycosylated by incubation with 0·15 U of peptide N-glycosidase F (Boehringer Mannheim) in 0·4 % ammonium bicarbonate, pH 8·5, at 37 °C for 16 h. Samples were fractionated on a prepacked cartridge Sep-Pak C18 (Waters); peptide fractions were collected manually.
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RESULTS |
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Validation of the PoSl primary structure and characterization of its glycoside moiety
Comparison of the PoSl amino acid sequence with that of the mature protein N-terminus allowed us to identify the putative PoSl pre-propeptide. The putative pre-peptide (124 108) was identified as a secretion signal by the program SignalP; consequently the propeptide was established as peptide (107 1).
The mature protein N-terminal sequence had previously been determined from the electroblotted purified protein (GPDDPALPPD...) (Palmieri et al., 2001). Edman degradation of the purified native protein actually gave rise to two equimolar PTH amino acids for each step. Other than the above N-terminal sequence, a second one (AQVPTLGTVFEL), corresponding to a polypeptide chain starting from A690 of PoSl, was detectable. Hence, it can be inferred that the F689-A690 peptide bond is hydrolysed in the mature protein and the cleaved peptide remains associated with the catalytic domain in a non-covalent complex, since no Cys residues in the cleaved peptide are present.
The PoSl amino acid sequence is characterized by seven putative glycosylation sites. As previously reported (Palmieri et al., 2001), MALDIMS analysis of permethylated N-linked sugar released by hydrolysis with N-glycosidase F showed the occurrence of several high mannose moieties with molecular mass ranging from 1580·0 and 2603·6 m/z and identified as Hex5HexNAc2 and Hex10HexNAc2. The other molecular ions at m/z 1784·0, 1987·5, 2191·5 and 2394·6 were identified as homologous structures having between three and six mannose residues linked to the pentasaccharide core.
To verify the deduced PoSl amino acid sequence, the PoSl protein was reduced, alkylated and then digested with trypsin, and the peptide mixture was directly analysed by MALDIMS, allowing the identification of several peptides (Fig. 3). This analysis also showed three different clusters of signals each characterized by a pattern of m/z values differing in 162 Da, in agreement with the presence of the high mannose glycosidic moiety, as described above. The assignments of the different molecular masses to the corresponding peptides (Table 2
) led to the identification of Asn106, Asn479 and Asn728 as N-glycosylation sites. As shown in Fig. 3
, the two residues Asn479 and Asn728 were present in both glycosylated and unglycosylated state. A further picture of PoSl N-glycosylation sites was obtained after deglycosylation of peptide mixture by N-glycosidase F. Indeed, new signals were detected by MALDIMS analysis at 3440·1, 5087·1, 5484·1 and 6361·2 m/z, corresponding to the expected molecular mass of peptides 707738, 438485, 59112 and 59120, respectively, each increased by 1 Da, since the N-glycosylated Asn residues are converted to Asp following N-glycosidase F treatment. Moreover, an additional peak at m/z 1793·0 was present after deglycosylation. This mass value is in agreement with the expected molecular mass of the peptide 628642 (containing the potential N-glycosylation site Asn628) increased by 1 Da. This result led to the assignment of Asn628 as an N-glycosylation site, even though the MALDIMS spectrum of the tryptic peptide mixture did not allow us to detect the cluster of m/z peaks related to the Asn628 glycoforms. Hence, two out of seven putative PoSl N-glycosylation sites were found to be glycosylated, Asn479 and Asn728 were found to be both glycosylated and unglycosylated, whilst Asn238 and Asn301 were only found unmodified. MALDIMS analyses did not allow mapping of the last putative N-glycosylation site, Asn335. This could be due to an incomplete extraction of the peptides from the gel or to the well-known MALDI suppression phenomena.
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Homology search analysis and pcsl cDNA cloning and sequencing
PoSl shows a high level of identity with a hypothetical protein from Neurospora crassa (38 %, BLAST E score=2·1x10121), a subtilisin-like serine protease from the fungus Metarhizium anisopliae, Pr1C (39 %, E score=2·0x10114), and a minor extracellular serine protease from Bacillus subtilis, Vpr (31 %, E score=7·1x1041).
Ph. chrysosporium is the only basidiomycete whose genome has been sequenced to date. BLAST analysis of this genome (http://genome.jgi-psf.org/whiterot1/whiterot1.home.html) resulted in the identification of two PoSl homologous sequences, about 112 kb from each other. One of these, designated pc.18.58.1, appeared to encode a protein which was very similar to PoSl. RNA was extracted from Ph. chrysosporium and specific cDNA was amplified using oligonucleotide pairs designed on the basis of the genomic sequence and protein homology. The amplified cDNA was longer than pc.18.58.1 at its 5' terminus, even if it did not contain the start translational codon. Moreover, the alignment between pc.18.58.1 and AJ748587 showed a 6 aa insertion in the former, due to an incorrect exon end. The deduced amino acid sequence corresponding to the amplified cDNA showed an identity of 65 % with PoSl. Fourteen out of 15 introns in the gene sequence were in the same positions as those of the posl gene (Fig. 4).
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DISCUSSION |
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The PoSl coding sequence was determined and the deduced protein primary structure confirmed by MALDIMS mapping. This approach has proven useful for the identification of the N-glycosylation sites and characterization of the glycoside moiety of the protein. PoSl is homologous to many other serine proteases, especially in the areas surrounding the amino acids that are known to be involved in the active site of subtilisin (D, H and S).
The first amino acid (G1) of the mature protein was identified, allowing determination of the PoSl pro-sequence. Pro-sequences are usually removed from the catalytic domain by self-digestion or by another protease upon completion of protein folding. The peptide bond K1G1, which connects the PoSl pro-sequence to its catalytic domain, should be cleaved by a protease with trypsin-like specificity different from the known PoSl substrate preference (Palmieri et al., 2001). Moreover, no significant activity of PoSl towards the chromogenic peptide D-Val-Leu-Lys-p-nitroanilide was observed (data not shown). On the other hand, PoSl has a D residue in the position corresponding to E156 in subtilisin BPN'. Subtilases with a negative charge on this residue are reported to have an additional ability to cleave after K at the P1 position (Gron et al., 1992
; Voorhorst et al., 1997
). Furthermore, all the interactions of the substrate P4P4' residue side chains with the S1 pocket can contribute to binding; thus it cannot be excluded that PoSl zymogen can undergo autolysis to self-remove the pro-peptide from unprocessed protein during the maturation process.
PoSl shows a high identity with hypothetical proteins from N. crassa and Trichoderma reesei, and Pr1C, a bacterial-type subtilisin-like serine protease from the fungus M. anisopliae. Eleven subtilisin-like proteins were identified in the latter fungus (Bagga et al., 2004), ten of which displayed identities ranging from 93 to 98 %, being classified as proteinase K-like subtilisins. The unique exception was the bacterial-type subtilisin Pr1C, which is largely divergent from the others. Among the bacterial subtilases, Vpr, a minor extracellular serine protease from B. subtilis (Sloma et al., 1991
), shows the highest identity with PoSl. This bacterial subtilase belongs to the pyrolysin family, according to the classification of Siezen & Leunissen (1997)
.
Interestingly, a Ph. chrysosporium genomic sequence encoding a hypothetical protein, PcSl, showing the highest identity with PoSl (65 %), has been identified and the corresponding cDNA was amplified and sequenced, thus demonstrating the expression of the pcsl gene in this fungus. The posl and pcsl genes shared 57 % of their sequences and their intron/exon structures were very similar (Fig. 4). Furthermore, all the glycosylated Asn identified in PoSl was located in consensus sequences conserved in PcSl, suggesting common structural features between the two proteins. Indeed, a 76 kDa protease, inhibited by PMSF, has previously been reported in Ph. chrysosporium culture broth. This enzyme is the most abundant protease produced in the presence of both excess and limiting nitrogen source (Dass et al., 1995
). These data suggest that this protease is the one encoded by the pcsl gene. Hence, a new subgroup of subtilisin-like proteases from ascomycete and basidiomycete fungi, belonging to the pyrolysin family, can be defined, including the proteases PoSl and PcSl from the basidiomycete fungi Pl. ostreatus and Ph. chrysosporium, the hypothetical protein from N. crassa, and Pr1C from M. anisopliae. In Fig. 5
the alignment of these protease sequences is shown.
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All the members of the new fungal pyrolysin subfamily have a long C-terminal extension, which is less conserved than the catalytic region (Siezen & Leunissen, 1997; Voorhorst et al., 1996
). Mature PoSl shows a cleaved peptide bond in this region, probably caused by an autocatalytic event, on the basis of its specificity. The cleaved peptide remains associated with the catalytic domain in a non-covalent complex, as demonstrated by the two N-terminal sequences obtained from the native protein and confirmed by the MALDIMS characterization of the protein. This finding suggests that the cleaved bond belongs to an exposed loop of the protein and that, after the proteolytic event, the newly generated peptide remains tightly bound to the core of the protein.
The occurrence of this class of proteases in a wide variety of fungal genera could be suggestive of the crucial physiological role played by these proteins in the micro-organisms which produce them. With regard to Pl. ostreatus, the role proposed for PoSl, a member of this subfamily, would be to start a cascade of proteolytic reactions, leading to the activation of other extracellular proteases.
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ACKNOWLEDGEMENTS |
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Received 29 June 2004;
revised 13 October 2004;
accepted 15 October 2004.
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