Processing of the Phalloidin Proprotein by Prolyl Oligopeptidase from the Mushroom Conocybe albipes

The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently. The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently. Deadly poisonous mushrooms in the genera Amanita, Galerina, Lepiota, and Conocybe synthesize the cyclic peptide amatoxins and phallotoxins (1.Brady L.R. Benedict R.G. Tyler V.E. Stuntz D.E. Malone M.H. Lloydia. 1975; 38: 172-173PubMed Google Scholar, 2.Enjalbert F. Cassanas G. Rapior S. Renault C. Chaumont J.-P. Mycologia. 2004; 96: 720-729Crossref PubMed Scopus (0) Google Scholar, 3.Hallen H.E. Watling R. Adams G.C. Mycol. Res. 2003; 107: 969-979Crossref PubMed Scopus (66) Google Scholar, 4.Wieland T. Peptides of Poisonous Amanita Mushrooms. Springer-Verlag New York Inc., New York1986Crossref Google Scholar). Structurally, amatoxins (e.g. α-amanitin) are bicyclic octapeptides, and phallotoxins (e.g. phalloidin and phallacidin) are chemically related bicyclic heptapeptides. Both are hydroxylated and contain a Trp-Cys cross-bridge (tryptathionine), which has not been found in other natural products (5.May J.P. Perrin D.M. Pept. Sci. 2007; 88: 714-724Crossref Scopus (35) Google Scholar). Phallotoxins also contain one d amino acid (d-hydroxy-Asp or d-Thr). Despite their chemical similarity, amatoxins and phallotoxins have different modes of action; amatoxins are specific inhibitors of RNA polymerase II, and phallotoxins bind and stabilize F-actin (6.Bushnell D.A. Cramer P. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1218-1222Crossref PubMed Scopus (247) Google Scholar, 7.Wulf E. Deboben A. Bautz F.A. Faulstich H. Wieland T. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4498-4502Crossref PubMed Scopus (623) Google Scholar). Unlike other known cyclic peptides from fungi, the amatoxins and phallotoxins are biosynthesized on ribosomes instead of nonribosomal peptide synthetases (8.Hallen H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Scholar). In A. bisporigera, the genes for α-amanitin and phallacidin are translated as 35 and 34-amino acid proproteins, respectively (8.Hallen H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Scholar). Although α-amanitin and phallacidin have only three amino acids in common, the upstream and downstream sequences of the proproteins are highly similar. Furthermore, the genome of A. bisporigera contains at least another 20 related sequences which collectively are characterized by conserved sequences flanking a hypervariable “toxin” region of 7–10 amino acids (8.Hallen H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Scholar) (Fig. 1B). We refer to this family of genes as the MSDIN family for the first five highly conserved amino acids of the proproteins. Apparently, these poisonous mushrooms have evolved a mechanism of combinatorial peptide biosynthesis that uses the same biochemical template to synthesize a wide range of small, cyclic peptides. Among the conserved amino acids in the MSDIN gene family are two invariant Pro residues flanking the hypervariable region (Fig. 1B). All known amatoxins and phallotoxins contain one Pro, and A. phalloides is also known to make other cyclic peptides (of 6–10 amino acids), all of which contain at least one Pro residue (4.Wieland T. Peptides of Poisonous Amanita Mushrooms. Springer-Verlag New York Inc., New York1986Crossref Google Scholar). Post-translational processing of the MSDIN proproteins is predicted to involve cleavage at these Pro residues, resulting in one Pro remaining in the toxin and the other being removed (Fig. 1B). The goal of the present work was to identify the enzyme that cleaves the MSDIN proproteins to release the peptides of the mature toxins. To identify the responsible peptidase, it was necessary to find a practical starting material. Most toxin-producing fungi in the genus Amanita are obligately ectomycorrhizal with their host plants, and they grow very slowly and do not produce toxins in culture. Galerina marginata produces toxins in culture but grows slowly (9.Muraoka S. Shinozawa T. J. Biosci. Bioeng. 2000; 89: 73-76Crossref PubMed Scopus (12) Google Scholar). Some species of the mushroom genus Conocybe produce phallotoxins or amatoxins (1.Brady L.R. Benedict R.G. Tyler V.E. Stuntz D.E. Malone M.H. Lloydia. 1975; 38: 172-173PubMed Google Scholar, 3.Hallen H.E. Watling R. Adams G.C. Mycol. Res. 2003; 107: 969-979Crossref PubMed Scopus (66) Google Scholar). Conocybe species are abundant locally at certain seasons. In particular, Conocybe albipes (Fig. 1C), which produces the phallotoxin phalloidin, grows on lawns in the Midwest United States, and in the spring it is possible to collect large (>1 kg) quantities (10.Hutchinson L.J. Madzia S.E. Barron G.L. Can. J. Bot. 1996; 74: 431-434Crossref Scopus (28) Google Scholar). For these reasons we chose C. albipes as a starting material for identification of the enzyme that processes the toxin proproteins. Here we report the purification of the enzyme that releases the mature heptapeptide from a phallotoxin precursor and show that it is a member of the prolyl oligopeptidase (POP) 2The abbreviations used are: POPprolyl oligopeptidaseZ-benzyloxycarbonylpNAp-nitroanilideLCliquid chromatographyMS/MStandem mass spectrometryRACErapid amplification of cDNA endsHPLChigh performance liquid chromatography. subfamily of serine proteases. prolyl oligopeptidase benzyloxycarbonyl p-nitroanilide liquid chromatography tandem mass spectrometry rapid amplification of cDNA ends high performance liquid chromatography. Fruiting bodies (basidiocarps) of C. albipes were collected from lawns on the campus of Michigan State University during the spring and summer of 2008 and stored at −80 °C until use. Fifty grams of frozen mushroom were ground in liquid nitrogen in a mortar and pestle. The ground powder was resuspended in 200 ml of 50 mm Tris·HCl, pH 8.0, containing 10 ml of protease inhibitor mixture solution. The inhibitor mixture solution (10 ml) contained five tablets of Complete Mini EDTA-free protease inhibitor (Roche Applied Science) and 5 ml of a protease inhibitor mixture (Sigma P2714) that had been dissolved in 10 ml of deionized water. The sample was centrifuged in a Sorvall GSA rotor at 10,200 × g for 10 min at 4 °C. Ammonium sulfate was added to the supernatant to 30% saturation (16.6 gm/100 ml). After stirring at 4 °C for 30 min, the sample was centrifuged (10,200 × g, 10 min, 4 °C). Ammonium sulfate was then added to 60% saturation (the addition of 18.4 gm/100 ml) and centrifuged again. Ammonium sulfate was added to the supernatant to 80% saturation (the addition of 13.1 gm/100 ml), and the pellet from this final spin was redissolved in 5 ml of 20 mm NaH2PO4, pH 7.0. Ammonium sulfate was added to a final concentration of 1.7 m, and the sample was filtered through a 0.22-μm Millex filter (Millipore). Five ml was applied to a TosoHaas phenyl 5PW hydrophobic interaction column (7.5 mm × 7.5 cm) and eluted with a 30 min gradient from 20 mm NaH2PO4 + 1.7 m ammonium sulfate, pH 7.0, to 20 mm NaH2PO4, pH 7.0. The flow rate was 1 ml/min, and 1-ml fractions were collected. Protease inhibitor mixture solution (50 μl) was added to each fraction. Fractions with activity (see below) were buffer-exchanged into 10 mm KH2PO4, pH 6.8, 0.02% sodium azide on an Econo-Pac 10DG column (Bio-Rad). The sample (5 ml) was then applied to a Bio-Rad CHT5-I hydroxyapatite column (10 mm × 6.4 cm) and eluted with a gradient from 10 mm KH2PO4, pH 6.8, 0.02% sodium azide to 0.35 m KH2PO4, pH 6.8, 0.02% sodium azide over 30 min at a flow rate of 1 ml/min. Protease inhibitor mixture solution (50 μl) was added to each fraction. Active fractions were pooled and buffer-exchanged into 25 mm Tris·HCl, pH 8.0. The sample (5 ml) was applied onto a TosoHaas DEAE-5PW column (7.5 mm × 7.5 cm) and eluted with a 40-min linear gradient from 25 mm Tris·HCl, pH 8.0, to 40% 25 mm Tris·HCl, pH 8.0, 0.6 m NaCl. Protease inhibitor mixture solution (50 μl) was added to each fraction. The chromogenic POP substrate Z-Gly-Pro-pNA was purchased from Sigma-Aldrich and used at a final concentration of 1 mm. The POP inhibitor Z-Pro-prolinal was purchased from Biomol (Plymouth Meeting, PA) and used at 1 μm. All assays were done at 37 °C. Synthetic peptides (Table 1) representing proproteins of phalloidin, phallacidin, and α-amanitin were obtained from Bachem, Celtek, or the Michigan State University Research Technology Support Facility. The peptides were dissolved in deionized water at 1 mg/ml. Purified POP (50 μl of a 1-ml fraction (typically in 25 mm Tris·HCl, pH 8.0) was mixed with 5 μl of synthetic peptide (7 μl for the phalloidin precursor). At the end of the incubation 1 μl of 100 mm DTT was added, and the samples held for another 10 min at 21 °C and then stored at −80 °C. POP was inactivated by heating at 100 °C for 2 min (boiled control).TABLE 1Synthetic peptides used in this studyNameSequencePhalloidin precursor (22-mer)MSDINATRLPAWLATCPCAGDDPhalloidin mature peptideAWLATCPPhallacidin precursor (17-mer)ATRLPAWLVDCPCVGDDPhallacidin mature peptideAWLVDCPα-Amanitin precursor (16-mer)TRLPIWGIGCNPCVGDα-Amanitin mature peptideIWGIGCNP Open table in a new tab Peptides were separated on an Agilent Model 1100 HPLC equipped with a C18 column (Vydac 218TP54). Elution solution A was water + 0.1% trifluoroacetic acid, and solution B was acetonitrile + 0.075% trifluoroacetic acid. Flow rate was 1 ml/min with a gradient from 20% solution A to 60% solution B in 15 min. Detection was at 280 nm. Peaks were collected manually, evaporated by vacuum centrifugation, and transferred to autosampler vials. LC-MS/MS was performed on a Micromass Q-TOF Ultima API instrument equipped with a Waters CapLC capillary HPLC. Spectra were analyzed with MassLynx software (Waters). MS/MS fragmentation of the most abundant molecule in each fraction was performed. The spectra were converted with the MaxEnt 3 module (MassLynx) into singly charged spectra. The spectra were then submitted to the PepSeq module of MassLynx for peptide sequence prediction. The molecular mass was set to be the mass shown on the spectrum. Minimum mass S.D. was set to 0.025. Sequence display threshold (% probability) was set to 1. Peptide identity was further confirmed with the Proteins module of MassLynx. The putative peptide sequences that matched the obtained mass spectra were used to calculate potential fragments. The MS/MS spectra of the peptides were then loaded and used to search against the fragments. were the the predicted sequences were to the peptide sequences. RNA was the RNA for and were synthesized 1 of RNA with and were on five predicted POP gene for A or B for or for or for or T. A was and analysis of sequence by to known POP was used as a template for the and were performed and from the were used to a cDNA of ml) of the fractions with POP activity were against 2 of deionized water and was performed on (Bio-Rad). were with were to A. 1996; PubMed Scopus Google and the peptides were with a Waters onto a Waters C18 peptide (5 × 20 at 4 in 0.1% acid. The peptides were then eluted onto a Waters C18 column 100 × 100 over 35 min with a gradient of B to B in 21 min, to B from min and to B at min a Waters A 0.1% B 0.1% The flow rate was to at 21 min and to at 35 min. peptides were into a mass with a The five in each were then to by and the resulting MS/MS spectra were converted to the instrument were the against a of the of from the to which the sequence of had been added D.M. J.S. PubMed Scopus Google Scholar). The was then analyzed to protein the A. E. R. 2003; PubMed Scopus Google Scholar). the filter were for all to 2 of of of peptide of MS/MS of peptide to a synthetic 22-mer peptide to the proprotein of phalloidin from A. bisporigera as substrate (Table we purified in three from C. albipes an enzyme of the heptapeptide to The cleavage enzyme was very to the of a mixture of protease inhibitors in all of the purification After the final (Fig. the cleavage activity as a by (Fig. The molecular mass of the purified protein was (Fig. At each of the activity against the chromogenic POP substrate Z-Gly-Pro-pNA and cleavage activity were not that the cleavage enzyme is a We a gene encoding a POP from C. albipes on putative POP genes from other and and were used to the sequence of the cDNA The C. albipes POP cDNA has a region of and a region to of The protein has amino acids and a molecular mass of a of and predicted peptide S. G. H. 2007; PubMed Scopus Google Scholar). has the conserved as as all of the amino acids of the not all residues of the are conserved PubMed Scopus Google Scholar, C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) (Fig. The sequence of amino acid identity to from the mushrooms and with of and identity of and The sequence of is also strongly to other putative or known from plants, and has 40% identity with POP PubMed Scopus Google Scholar). of were found in all at the of the and A. bisporigera (8.Hallen H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Scholar). The purified cleavage enzyme (Fig. was with and to The were against a in of to which was added the sequence of peptides that matched were obtained (Fig. The was R. 2004; PubMed Scopus Google Scholar). The in the was one peptide and a of the cleavage enzyme as the of A of cleavage of the synthetic phalloidin peptide is shown in Peaks were by mass of the and by of the Mass spectra of the intermediate at min and the at min are shown in A and All peptide sequences were confirmed by MS/MS the PepSeq module of MassLynx. Peptide were further confirmed with the Proteins module of of the at min in 4 of the at min in 4 mature The sequence of the final indicates that the C. albipes enzyme on the end of Pro, other the cleavage of the synthetic proprotein to the final only one intermediate was which of the N-terminal peptide by cleavage at the C-terminal in this and other found for the C-terminal peptide cleavage the N-terminal Pro was also for the of the intermediate or the final of the products during the indicates that the same POP enzyme cleaves at Pro residues of the phalloidin precursor and that the enzyme the C-terminal Pro bond the N-terminal Pro bond (Fig. the enzyme cleaves but cleaves the C-terminal Pro bond much faster than the The of an intermediate indicates that not the intermediate is from the enzyme cleavage 5 and The amino acid sequence of phalloidin from that of phallacidin by two amino A synthetic peptide on phallacidin was also cleaved by (Fig. A and The specific POP inhibitor Z-Pro-prolinal cleavage of the phallacidin precursor peptide (Fig. the inhibitor also cleavage of the phalloidin precursor not the same of and a peptide on the proprotein of α-amanitin was cleaved than the phallacidin precursor (Fig. 2 only a of the mature amanitin had (Fig. The mass of the mature amanitin cleavage eluted at min, is shown in The sequences of the proproteins of and other of the MSDIN gene family that a Pro-specific peptidase the initial post-translational processing of this of natural products (8.Hallen H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Scholar). biochemical and molecular were used to in the phalloidin-producing mushroom C. albipes, the initial processing is by a member of the POP subfamily of serine proteases (EC family The same enzyme cleaves at the of flanking Pro residues to release the peptide of the mature The enzyme cleaves or much at the C-terminal Pro and is by accumulation of the N-terminal or sequences encoding have been in plants, and other in the All have at least one gene strongly to and to but on a of than 20 this to POP have been in they have been shown to of peptides peptide S. 2007; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, 2007; PubMed Scopus Google Scholar, J. C. Sci. 2007; PubMed Scopus Google Scholar, Sci. 2002; PubMed Scopus Google Scholar, Pept. Sci. PubMed Scopus Google Scholar). in of POP have been with and to PubMed Scopus (28) Google Scholar). A POP inhibitor in S. 2007; PubMed Scopus Google Scholar). are also being as a for an by peptides in certain C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Pept. Sci. PubMed Scopus Google Scholar). The a of which is to the precursor proproteins of the cyclic peptides of and and peptides synthesized on ribosomes are known from plants, and 2007; Google Scholar, P. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, D.A. S. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). the toxins of Amanita species and other these peptides are synthesized as proproteins at least one post-translational The toxins are cleaved at amino acids and and only one cleavage is necessary to release the mature G. J.D. J. J. 2003; PubMed Scopus Google Scholar). The of the and other which are cyclic peptides made by be cleaved to release the mature and this is predicted to be by an of the family that is the D.A. S. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The mechanism of of the is not that it be A. PubMed Scopus Google Scholar). The are cyclic peptides that are amino than the mushroom toxins. are cleaved from their proproteins by an which 2007; Google Scholar, R.G. J. 2007; PubMed Scopus (184) Google Scholar). In we of phalloidin heptapeptide or amanitin and of the MSDIN family of is not by In the biosynthesis of which is a cyclic the precursor proprotein be cleaved to release the mature and are by two and and the processing S. S. C. J. S. 2007; PubMed Scopus Google Scholar). family of small, cyclic the peptides of which are are by a E. PubMed Scopus Google Scholar, R. J. G. J. PubMed Scopus Google Scholar). In being by a Pro-specific peptidase, processing of the MSDIN peptides of poisonous mushrooms by a different mechanism than is known from on other that the of POP is by than the presence of an Pro POP has been to at other amino as or 2007; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). do not at all possible Pro residues 2007; PubMed Scopus Google Scholar, A. J. 104: PubMed Scopus Google Scholar). For POP of peptides a Pro but not Pro residues R. J. PubMed Google Scholar). Some of the MSDIN family have predicted Pro residues the hypervariable region H.E. Luo H. Scott-Craig J.S. Walton J.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19097-19101Crossref PubMed Scopus (184) Google Furthermore, cyclic peptides purified from A. phalloides have Pro residues, as and (4.Wieland T. Peptides of Poisonous Amanita Mushrooms. Springer-Verlag New York Inc., New York1986Crossref Google Scholar). as these are from their proproteins the amatoxins and then the responsible POP not at all possible Pro is found with the cyclic also made by A. which contains Pro residues in two (4.Wieland T. Peptides of Poisonous Amanita Mushrooms. Springer-Verlag New York Inc., New York1986Crossref Google Scholar). that cleaves two phallotoxin than an α-amanitin precursor (Fig. be related to the that C. albipes only produces it be that this is to different of the synthetic peptides used (Table The amino acids of the phalloidin precursor with the phallacidin and α-amanitin (Table 1) amino acids to interaction with with synthetic of the MSDIN family and with the proproteins be necessary to the substrate of and other from other toxin-producing T. J. 104: PubMed Scopus Google Scholar) found POP activity in of species of mushroom and of the species that be POP activity was the same purified an POP from the mushroom T. J. 107: PubMed Scopus Google Scholar). In was very to during which is of also that is which is with a in processing of the phalloidin do that is the enzyme that processes the phalloidin precursor in The of POP in toxin mushrooms is an in the of A. a in Although as be cleaved by in the in of in plants, and are in most S. 2007; PubMed Scopus Google Scholar). In in this we have shown that the POP of the phalloidin-producing mushroom C. albipes and the precursor of phalloidin, the heptapeptide of the mature in the biosynthesis of phalloidin of to and of the We and the Michigan State University Research Technology Support for of and We also for with