Protein Sequence Analysis, Cloning, and Expression of Flammutoxin, a Pore-forming Cytolysin from Flammulina velutipes MATURATION OF DIMERIC PRECURSOR TO MONOMERIC ACTIVE FORM BY CARBOXYL-TERMINAL TRUNCATION*

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes , is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa

Pore-forming cytolytic proteins are distributed in a wide variety of eukaryotic and prokaryotic organisms (1,2). Complement, perforin from the cytotoxic T-cells, ␣-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, aerolysin from Aeromonas hydrophila, and some others have been intensively studied in terms of pathophysiological functions (2)(3)(4)(5)(6). The self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly, membrane insertion, and molecular architecture of transmembrane pores (3)(4)(5)(6). Several cytolytic proteins have been isolated from the basidiocarps of both toxic and edible mushrooms, and their pore-forming properties as well as cardiotoxicity and cytotoxicity were studied (7)(8)(9)(10). Although the physiological function of the mushroom cytolysins remains enigmatic, recent studies have implied the involvement of hemolytic proteins in the fruiting initiation of some mushrooms. The Aa-Pri1 gene, which encodes a putative 16-kDa protein, has been shown to be specifically expressed in the fruiting initiation of the edible mushroom Agrocybe aegerita (11). Aegerolysin was isolated as a 17-kDa hemolytic protein from the basidiocarps of A. aegerita, and it was preferentially detected in the primordia and immature fruiting bodies of the mushroom (12).
Lin et al. (9) isolated a cardiotoxic and cytolytic 22-kDa protein from the basidiocarps of the edible mushroom Flammulina velutipes, and designated it flammutoxin (FTX). 1 Later, Bernheimer and Oppenheim (13) purified a hemolytic protein of 32 kDa from the same mushroom and referred to it as FTX on the assumption that the FTX of Lin et al. (9) derived from their 32-kDa FTX by partial proteolysis. We isolated FTX as a 31-kDa single hemolysin of F. velutipes, determined the Nterminal 28 residues, and studied the molecular basis of the cytolytic action of the protein (14). Our results showed that FTX assembles into a ring-shaped oligomer with outer and inner diameters of 10 and 5 nm, respectively, which forms membrane pores with a functional diameter of 4 -5 nm and causes an osmotic burst of human erythrocytes (14). By using planar lipid bilayers, we showed that FTX forms a cation-selective, voltage-gated channel with a diameter of 4 -5 nm (15).
Watanabe et al. (16) purified a 30-kDa transepithelial electrical resistance-decreasing protein from the basidiocarps of F. velutipes, which increased tight junctional permeability of human intestinal Caco-2 monolayers. The N-terminal amino acid sequence of the purified protein was identical with that of FTX reported by us (14,16). Watanabe et al. (16) cloned a cDNA encoding a 272-residue protein (AB012289) and concluded that the cloned cDNA encodes the transepithelial electrical resistance-decreasing protein, because N-terminal sequence and molecular mass of the predicted protein coincided with those of the purified protein (16). However, they did not express the cloned cDNA. Concurrently with their cloning, we cloned a cDNA encoding the same protein (GenBank TM accession number AB015948) and expressed the cDNA in Escherichia coli but failed to obtain hemolytically active recombinant protein.
Taken together with the fact that F. velutipes produces multiple FTX family proteins with N-terminal sequences similar to that of FTX (described below), it remained uncertain that the cloned cDNAs encode FTX or the transepithelial electrical resistance-decreasing protein. This prompted us to determine the complete primary structure of FTX by protein sequence analysis.
Sakamoto et al. (17) studied expression of genes in different developmental stages of F. velutipes and cloned C1 cDNA (Gen-Bank TM accession number AB030006), which was specifically expressed during the fruiting body formation. A search on the DDBJ/GenBank TM /EBI nucleotide sequence data bases indicated that the C1 cDNA is identical with the cDNAs cloned by us and by Watanabe et al. (16). The same group also analyzed chronological expression of proteins in F. velutipes by using twodimensional electrophoresis and showed that four 30 -32-kDa proteins, which had N-terminal sequences similar to that of FTX, were abundantly expressed in the fruiting bodies of F. velutipes (17). Furthermore, cDNAs identical with FTX cDNA were cloned from Hericium erinaceum, Agrocybe chaxingu, Pleurotus eryngi var. ferulae, Coprinus comatus, and Ganoderma lucidum, which cover different families of Basidiomycetes (GenBank TM accession numbers AY281063-AY281067). Thus, FTX and/or FTX family proteins are produced by F. velutipes and other mushrooms during the fruiting body formation.
In this study, we determined the complete primary structure of FTX by protein sequence analysis. As a result, protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Based on the sequence information obtained, we constructed expression systems for production of rFTXs of precursor and mature forms and studied the maturation process of FTX.

EXPERIMENTAL PROCEDURES
Materials-Basidiocarps of F. velutipes were purchased from the producers in Gunma and Miyagi Prefecture and stored at Ϫ40°C. The sources of other materials and chemicals used were as follows: N-tosyl-L-phenylalanyl-chloromethylketone (TPCK)-treated trypsin from Cooper Biomedical (Malvern, PA); S. aureus V8 protease from ICN Biomedicals (Costa Mesa, CA); endoproteinase Asp-N from Roche Applied Science; cyanogen bromide and tri-n-butyl phosphine from Wako Pure Chemical (Osaka, Japan); hydroxylamine HCl from Kanto Chemical (Tokyo, Japan); and 4-vinylpyridine from Tokyo Kasei (Tokyo, Japan). Achromobacter protease I was kindly supplied by Dr. T. Masaki of Ibaraki University (Ibaraki, Japan). Other chemicals used were of analytical grade.
Purification and S-Alkylation of FTX-FTX was purified from the basidiocarps of F. velutipes as described (14). FTX was reduced and pyridylethylated (PE) as described (18).
Chemical Cleavages-Methionyl bonds of PE-FTX were cleaved according to the method of Gross (19) with 1% (w/v) CNBr in 70% (v/v) formic acid. Asparaginyl-glycine bonds of PE-FTX were cleaved with 2 M hydroxylamine, as described by Bornstein and Balian (20). Aspartylproline bonds of PE-FTX were hydrolyzed in 1% (v/v) formic acid at 40°C for 72.5 h, according of the method of Landon (21). T7k peptide was hydrolyzed in 12 M HCl at room temperature for 15 h, as described by Titani and Narita (22).
Enzymatic Cleavages-PE-FTX was digested with Achromobacter protease I in 50 mM Tris-HCl buffer (pH 9.0) containing 2 M urea at 37°C for 18 h at an enzyme/substrate molar ratio of 1:100. PE-FTX was digested with S. aureus V8 protease at 37°C for 18 h in 50 mM sodium phosphate buffer (pH 7.8) containing 1.5 M urea and 2 mM EDTA at an enzyme/substrate molar ratio of 1:30. FTX was digested with TPCKtreated trypsin at 37°C for 18 h in 50 mM Tris-HCl buffer (pH 8.3) containing 4 M urea and 10 mM CaCl 2 at an enzyme/substrate molar ratio of 1:10. T7k peptide was digested by S. aureus V8 protease or thermolysin at 37°C for 18 h in 100 mM ammonium bicarbonate buffer (pH 7.8 or 8.2) containing 2 mM EDTA or 2 M urea plus 5 mM CaCl 2 , respectively, at an enzyme/substrate molar ratio of 1:10. M2 peptide was cleaved with endoproteinase Asp-N at 37°C for 18 h in 50 mM sodium phosphate buffer (pH 8.0) containing 2 M urea at an enzyme/ substrate weight ratio of 1:50.
Separation of Peptides-Peptides generated by enzymatic and chemical cleavages were separated by gel permeation chromatography (GPC) on tandem columns of TSKgel G2000SW XL and TSKgel G3000SW XL (7.8 ϫ 300 mm each; Tosoh, Tokyo) using a Gilson model 302 pump and a Hewlett Packard HP 1040M diode array detection system. Elution was conducted with 10 mM phosphate buffer (pH 6.0) containing 6 M guanidine HCl at a flow rate of 0.4 ml/min, and the effluent was monitored at 215, 260, 275, and 290 nm. The peptide fractions obtained were passed through a Sephadex G-25 fine column and were further separated by reverse phase high performance liquid chromatography (RP-HPLC) using a Gilson HPLC system or a Hewlett Packard model 1090M liquid chromatograph on an Aquapore RP-300 (4.6 ϫ 100 or 4.6 ϫ 100 mm; Applied Biosystems, Foster City, CA), an Aquapore PH-300 (2.1 ϫ 30 mm; Applied Biosystems), a TSKgel super ODS column (2.0 ϫ 48 mm; Tosoh), or a Nova-Pak C18 column (3.9 ϫ 300 mm; Waters Co., Milford, MA). Peptides were eluted with a linear gradient of acetonitrile (0 -80%) in 0.09% (v/v) trifluoroacetic acid at a flow rate of 0.5 or 0.2 ml/min.
Amino Acid Composition and Amino Acid Sequence Analyses-Compositional analyses were performed by precolumn derivatization with a Waters Pico-Tag system (23). The samples (50 -100 pmol) were hydrolyzed in vapor phase of 6 M HCl containing 0.1% (w/v) phenol at 110°C for 20 h. Automated Edman degradation was performed with an Applied Biosystem model 477A or model 493A protein sequencer (Applied Biosystems, Foster City, CA).
Mass Spectrometry-Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for proteins and peptides was performed on REFLEX (Bruker Daltonics, Bremen, Germany) with sinapinic acid or ␣-cyano-4-hydroxycinnamic acid as a matrix.
Nomenclature of Peptides-Peptides were abbreviated by a serial number prefixed with letter(s). The letters indicate the type of digestion as follows: M, cyanogen bromide; K, Achromobacter protease I; T, trypsin; E, S. aureus V8 protease; D, endoproteinase Asp-N; Th, thermolysin; NG, hydroxylamine; DP, 1% (v/v) formic acid. Secondary fragmentation products are indicated by hyphenation. The numbers in the peptide abbreviation do not correspond to the order of elution in HPLC but rather to their relative positions in the protein sequence, starting from the N terminus.
Amplification of the FTX cDNA by Reverse Transcription-PCR-Total RNAs were isolated from the basidiocarps of F. velutipes using the RNAgents total RNA isolation system (Promega, Madison, WI) and were used as the templates. The sense primers (i.e. 5Ј-ATGGATCCI-CARGTIAARACITCITGGGARGAYYT-3Ј, where I, R, and Y indicate inosine, G/A, and T/C, respectively, and the underline indicates the BamHI site) were synthesized based on the N-terminal amino acid sequence of FTX (14). The antisense primer was 5Ј-GCAAGCTT-TTTTTTTTTTTTTTTTTTTTTT-3Ј, where the underline indicates a HindIII site. After a reverse transcription using the oligo(dT) primer, 35 cycles of PCR were performed using Taq polymerase (TaKaRa, Kyoto, Japan). Amplified fragments of ϳ1 kbp were inserted into the SmaI site of pUC118 and sequenced. The resultant plasmid was designated pUF1.
Amplification of the 5Ј-End of the FTX cDNA by Reverse Transcription-PCR-After the reverse transcription of the total RNAs using the oligo(dT) primer described above, oligo(dA) was added to the 3Ј-ends of the reverse transcripts by using terminal deoxyribonucleotidyl transferase (TaKaRa). The 5Ј-end of the FTX cDNA was amplified by a PCR using the oligo(dT) primer and an antisense primer (i.e. 5Ј-CGCTCAAT-GGAAACTATCTCACGA-3Ј, which corresponds to the segment from the 469th to the 498th nucleotide of the FTX cDNA; Fig. 7). Amplified DNA fragments of ϳ600 bp were cloned into the SmaI site of pUC118 and sequenced. The resultant plasmid was designated pUF-N.
Construction of Expression Plasmids-The FTX cDNA was inserted into the NcoI site of pTrc99A (Amersham Biosciences) to produce rFTX with the same N-terminal sequence as that of FTX. The pUF1 was digested with EcoRI and HindIII, the resultant EcoRI-HindIII fragments were inserted into pTrc99A, and the cloned pTrc99A was designated pTF3. The pTF3 was digested with NcoI, blunted, and digested with SalI. To amplify the DNA segment from the 4th to the 413th nucleotide of the FTX cDNA (which corresponds to the N-terminal 135 amino acid residues of FTX; the nucleotide numbering is according to Fig. 7), a PCR was performed using the pUF-N as the template and the following primers. The sense primer was 5Ј-CCTCAAGTCAAGA-CAAGTTGGGAGGATCTC-3Ј, which corresponds to the N-terminal 10 amino acid residues of FTX (Fig. 7), and the antisense primer was 5Ј-GGTGTCGACTCCGTAGAAATCGAAATCTCG-3Ј, corresponding to the segment from the 387th to the 413th nucleotide (where the underline indicates a SalI site and nucleotide numbering is according to Fig.  7). Amplified DNA fragments of ϳ400 bp were digested with SalI and ligated with the SalI-digested pTF3 possessing the blunt end of the NcoI site. The resultant plasmid for expression of FTX precursor was designated pFTX272.
To construct an expression system for mature FTX, the DNA segment, which contained the FTX cDNA fragment from the 337th to the 756th nucleotide and a stop codon, was amplified by a PCR using pFTX272 as the template and the following primers. The sense primer was 5Ј-ATTCTGCAGTTGAGTCAGTCGATCACC-3Ј (where the underline indicates the PstI site and the segment from the 337th to the 360th nucleotide is included; the nucleotide numbering is according to Fig. 6), and the antisense primer was 5Ј-CTCAAGCTTTCACTTCACCGT-CAAAGGGGCAG-3Ј (where the underline and the double underline indicate the HindIII site and a stop codon, respectively, followed by the segment corresponding to the 737th to the 756th nucleotide). Amplified DNA fragments of ϳ400 bp were digested with PstI and HindIII and ligated with the double-digested pFTX272 with PstI and HindIII. The resultant plasmid was designated pFTX252.
DNA Sequencing-The cycle-sequencing reaction was performed with Sequi Therm Long Read cycle sequencing kits containing M13 forward and reverse IR-dyeprimers (Epicenter Technologies, Madison, WI). A Long Read IR DNA sequencing system (Li-Cor model 4000L; Li-Cor Inc., Lincoln, NE) was used for sequencing. The resultant sequences were analyzed using the GENETYX software package. A similarity search for nucleotide sequences was performed on the DDBJ/ GenBank TM /EBI nucleotide sequence databases.
Expression, Renaturation, and Purification of rFTX-E. coli DH5␣ cells harboring pFTX272 or pFTX252 were grown at 37°C in 2ϫ YT medium (1.6% Bacto-Trypton, 1% Bacto-Yeast extract, and 0.5% NaCl; Difco) with ampicillin (100 g/ml). When optical density at 660 nm of the culture reached 0.4, isopropyl-␤-D-thiogalactoside was added at 1 mM. After further cultivation for 4 h, bacteria were collected by centrifugation and suspended in 20 mM sodium phosphate buffer (pH 7.2) containing 20 mM EDTA and 1 mM phenylmethylsulfonyl chloride. The suspension was passed through a French pressure cell (SLM Instruments Inc., Rochester, NY) at 1200 kg/cm 2 and was centrifuged at 14,000 ϫ g at 4°C for 20 min. The precipitates obtained were suspended in 20 mM sodium phosphate buffer (pH 7.2), containing 4% (w/v) Triton X-100 and 20 mM EDTA, and incubated at room temperature for 12 h. After centrifugation at 14,000 ϫ g for 20 min, the white precipitates obtained were dissolved in 50 mM Tris-HCl buffer (pH 8.5) containing 8 M urea and incubated at 25°C for 1 h. Proteins were refolded at 4°C by the stepwise dialysis against 10 mM Tris-HCl buffer (pH 8.5) containing 4, 2, 1, 0.5, or 0 M urea and were loaded onto a TSKgel DEAE-5PW column (7.5 ϫ 200 mm; Tosoh). Adsorbed proteins were eluted with a linear gradient of NaCl (0 -300 mM). The FTX fraction, eluted with 120 -150 mM NaCl, was mixed with the same volume of 10 mM sodium phosphate buffer (pH 7.2) containing ammonium sulfate (40% saturation) and loaded onto a TSKgel Phenyl-5PW column (7.5 ϫ 200 mm; Tosoh). Adsorbed proteins were eluted with a linear gradient of ammonium sulfate (20 to 0% saturation). rFTX251 (i.e. rFTX without the C-terminal 20 residues) was assayed for its hemolytic activity toward human erythrocytes, whereas rFTX271 (i.e. rFTX with the C-terminal 20 residues) was assayed by Western immunoblotting using anti-FTX serum. Protein concentration was assayed as described by Bradford, using bovine serum albumin as a standard (24).
Hemolytic Assay-Hemolytic assay was performed as described (14). Human erythrocytes (3 ϫ 10 7 cells/ml) were incubated with rFTX (the final concentrations 0.1-100 g/ml) at 25°C for 30 min. After centrifugation at 600 ϫ g for 5 min, the supernatants obtained were assayed for absorbance at 541 nm. 100% lysis was defined as the absorbance of the supernatants obtained from the osmotically lysed cells. One hemo-lytic unit was defined as the amount of FTX, which caused 50% hemolysis under the conditions described.
Cross-linking of rFTX with Glutaraldehyde-rFTX271 or rFTX251 (final concentration of each protein 5.0 or 4.6 g/ml, respectively) was treated with 0.05% (w/v) glutaraldehyde at 20°C for 20 min as described (25). The glutaraldehyde-treated rFTX was heated in the presence of 2% (w/v) SDS and 5% 2-mercaptoethanol at 100°C for 5 min and subjected to Western immunoblotting using anti-FTX serum as described (14).
Assembly of rFTX into Membrane Pore Complex-Complex formation by rFTX was assayed as described (14). rFTX (0.5-2.0 g) was incubated with human erythrocytes (1.0 ϫ 10 8 cells) in 1 ml of Tris-buffered saline at 25°C for 30 min. The erythrocytes were washed twice with 5 mM Tris-HCl buffer, pH 7.2, and the erythrocyte membranes obtained were solubilized in 2% (w/v) SDS at 25°C for 5 min. The solubilized membranes were subjected to Western immunoblotting using anti-FTX serum.
Isolation and Electron Microscopy of Pore Complexes-Pore complexes of rFTX were isolated and analyzed by electron microscopy as described (10,14). Human erythrocytes (3 ϫ 10 9 cells) were incubated with rFTX271 or rFTX251 (50 g of each) in 50 ml of Tris-buffered saline at 25°C for 30 min. Erythrocyte membranes were collected and solubilized with 2% SDS (w/v) at 25°C and loaded onto a 10 -40% (w/w) sucrose gradient in 10 mM Tris-HCl buffer (pH 7.2) containing 0.1% SDS. Centrifugation was performed using a Beckman SW40Ti rotor at 32,000 rpm for 19 h at 4°C. Fractions were analyzed by Western immunoblotting using anti-FTX serum. Fractions containing the pore complexes were stained negatively with 1% (w/v) sodium phosphotungstic acid (pH 7.2) and examined under an electron microscope H-8100 (Hitachi, Tokyo, Japan) at an acceleration voltage of 80 kV.
Trypsin Digestion of rFTX271-rFTX271 (100 g/ml) was treated with TPCK-treated trypsin (10 g/ml) in 20 mM Tris-HCl buffer (pH 8.0) at 37°C for 0 -24 h. Small portions were withdrawn and mixed with soybean trypsin inhibitor (final concentration, 100 g/ml) and were subjected to SDS-PAGE or hemolytic assay. For MALDI-TOF MS, rFTX271 (28 g/ml) was treated with TPCK-treated trypsin (0.4 g/ml) at 37°C for 4 or 24 h, and small portions were immediately withdrawn and analyzed as described above.

RESULTS
Protein Sequence Analysis of FTX-The strategy used for determination of the complete amino acid sequence of FTX is summarized in Fig. 1. The complete sequence was established on the sequence information of the peptides generated by enzymatic and chemical cleavages of PE-FTX, together with the results of the amino acid compositions and molecular masses of the peptides. The N-terminal 72 residues and the C-terminal 107 residues (i.e. ϳ70% of the whole sequence) were sequenced by automated Edman degradation of intact PE-FTX and the fragments arising from PE-FTX by enzymatic digestions with Achromobacter protease I or S. aureus V8 protease and chemical cleavages with CNBr, hydroxylamine, or 1% formic acid (or at methionyl, Asn-Gly, or Asp-Pro bonds). The central part of FTX was completed by analyses of subdigest peptides derived from a surface-adhesive 7-kDa tryptic peptide (T7k) or a CNBr fragment (M2) by digestion with thermolysin, S. aureus V8 protease, or endoproteinase Asp-N. Some overlaps were provided by peptides obtained from T7k by 12 M HCl treatment. The molecular masses of FTX and several selected peptides were determined by MALDI-TOF MS to confirm the sequences obtained by automated Edman degradation.
CNBr Cleavage of PE-FTX-Cyanogen bromide fragments arising from PE-FTX (13 nmol) were first fractionated by GPC using tandem columns of two TSKgel G2000SW XL ( Fig. 2A), and the fractions I, II, and III obtained were further fractionated by RP-HPLC on an Aquapore RP-300 column. Fragments M1/M6 and M3/M4/M5 were isolated from the fractions I and III, respectively (Fig. 2, B and C). Fragment M2 was desalted with a column of Sephadex G-25 fine. Isolated fragments were analyzed for their compositions and sequences ( Fig. 1 and Table I). Fragments M2, M3, M4, M5, and M6 yielded 20-, 11-, 13-, 12-, and 27-residue sequences, respectively, and M6 was suggested to be the C-terminal fragment because of the lack of homoserine (Table I). The amino acid sequence of M2 overlapped with that of intact PE-FTX (Fig. 1), suggesting that M2 spans the central part of FTX based on the molecular size (i.e. ϳ15 kDa).
Digestion of PE-FTX with Achromobacter Protease I or S. aureus V8 -As shown in Fig. 2D, nine peptides, K1-K9, were resolved from an Achromobacter protease I digest of PE-FTX (4.5 nmol) by RP-HPLC on an Aquapore RP-300 column. Sequence analysis of peptide K4 revealed a new 26-residue sequence. K5 extended the sequence of M3 and overlapped with M4. Peptide K6 provided overlaps M4 -M6. The peptide K9 overlapped with M6 and extended the sequence ( Fig. 1 and Table II).
A digest of PE-FTX (4.6 nmol) by S. aureus V8 protease was fractionated by GPC using a TSKgel G2000SW XL column (Fig.  3A), and the fractions I-V obtained were further fractionated by RP-HPLC on an Aquapore RP-300 column (Fig. 3B and results not shown). Eight peptides, E1-E8, were isolated by RP-HPLC and were subjected to compositional and sequential analyses. As shown in Fig. 1, the E6 peptide obtained from the fraction II overlapped with the peptides K4 and K5, and the other peptides were assigned to the N-and C-terminal regions already established as above.
Cleavage of PE-FTX with Hydroxylamine or 1% Formic Acid-Because the M2 peptide arising from PE-FTX by the cleavage with CNBr contains an Asn-Gly bond (Fig. 1), PE-FTX (6.2 nmol) was cleaved with hydroxylamine to obtain a peptide starting from Gly 43 . The cleaved sample was fractionated by GPC using tandem columns of two TSKgel G2000SW XL and one TSKgel G3000SW XL , and three fractions (i.e. NG0, NG1, and NG2) were obtained (Fig. 3C). The apparent molecular masses of the fractions suggested that NG1 would be the peptide with the N-terminal Gly 43 , whereas NG0 and NG2 would be the intact molecule and an N-terminal fragment of FTX, respectively. The NG1 fragment was desalted with a column of Sephadex G-25 fine. An automated Edman degradation of the NG1 yielded the 22-residue sequence starting from the Gly 43 , which overlapped with the sequence of M2 (Fig. 1).
Because the NG1 peptide contains an acid-labile Asp-Pro bond (Fig. 1), PE-FTX (6.4 nmol) was cleaved with 1% formic acid to obtain a peptide starting from Pro 60 . The cleaved sample was fractionated by GPC using tandem columns of two TSKgel G2000SW XL and one TSKgel G3000SW XL , and three fractions were obtained (Fig. 3D). The apparent molecular masses of the fractions suggested that DP1 would be the peptide starting from Pro 60 , whereas DP0 and DP2 would be the intact molecule and an N-terminal fragment of FTX, respec- tively. The DP1 fragment was passed through a column of Sephadex G-25 fine and subjected to automated Edman degradation. As a result, the DP1 peptide gave the 25-residue sequence starting from Pro 60 (although there was some ambiguity in the sequence; Fig. 1).
Isolation of a Surface-adhesive Peptide T7k from the Central Part of FTX and Its Fragmentation with Thermolysin, S. aureus V8 Protease, or 12 M HCl-Since no peptide representing the central part of FTX was recovered from any proteolytic digest, those missing peptides must be lost by adsorption during the digestion or separation. To test these possibilities, PE-FTX was digested with trypsin, and the supernatant was subjected to GPC using tandem columns of two TSKgel G2000SW XL and one TSKgel G3000SW XL . Again, no large (Ͼ3-kDa) fragment was observed (results not shown). From the amino acid compositions of FTX and the sequences already placed, only a few trypsin-cleavable residues were left unsequenced. The tube was washed with 6 M guanidine hydrochloride and applied to the same column, and this time a double peak at 7-8 kDa appeared (Fig. 4A). The N-terminal sequences of these two fragments (both having Thr-Thr-Glu-Thr-Val-Trp-Ser-Tyr-Asp-Asn-Ser-Gln-) overlapped with that of DP1 and proved that they are the missing peptides. By MALDI-TOF MS using ␣-cyano-4-hydroxycinnamic acid as a matrix, mass (M ϩ H) ϩ of T7k was estimated to be 7656 Da (results not shown).
To fill the gap left between T7k and K4, M2 peptide (3 nmol) was digested with endoproteinase Asp-N. The digest was fractionated by GPC on a TSKgel G2000SW XL column, followed by RP-HPLC using a Nova-Pak C18 column (results not shown). Analyses for amino acid compositions and sequences indicated that fraction 20 contained the peptide, Asp-Ser-Lys-Thr-Lys-Ser-Lys-Glu-His-Thr-Leu-Thr-Asn-Thr-Trp, which overlapped Results are expressed as residues per peptide by amino acid (aa) analysis or, in parentheses, from the sequence (Fig. 1

TABLE II Amino acid compositions of Achromobacter protease I peptides (K1-K9)
Results are expressed as residues per peptide by amino acid (aa) analysis or, in parentheses, from the sequence (Fig. 1 with the C terminus of T7k and K4, resulting in the completion of the 251-residue sequence (Fig. 1). molecules purified from mushroom had heterogeneous C-terminal residues.

Molecular Mass of FTX and Heterogeneity of Its C Terminus-The
Expression of rFTX of Precursor and Mature Forms and Their Molecular Properties-As described under "Experimental Procedures," we amplified an ϳ1-kbp DNA by reverse transcription-PCR using a sense primer corresponding to the Nterminal 10 residues of FTX and an antisense oligo(dT) primer and analyzed the 5Ј-end of the cloned cDNA by the 5Ј-rapid amplification of cDNA end. The cloned cDNA consisted of 819 nucleotides, encoding 272 amino acid residues ( Fig. 6; Gen-Bank TM accession number AB015948). Based on the sequence determined by Edman degradation, we concluded that the cloned cDNA encodes a FTX precursor with the initial Met and additional C-terminal 20 residues (Figs. 1 and 6). Therefore, we constructed expression systems to produce rFTX of the precursor and mature forms. Briefly, the FTX cDNA was inserted into the NcoI site of pTrc99A to construct pFTX272, and the PstI-HindIII segment of pFTX272 was replaced with the DNA fragment corresponding to the C-terminal region of FTX to construct pFTX252 (Fig. 7A). Both the precursor and mature forms of rFTX were expressed as insoluble proteins in E. coli DH5␣, and no hemolytic activity was detected in the bacterial lysates. The insoluble rFTX fraction was unfolded in 8 M urea and refolded by a stepwise dialysis against 10 mM Tris-HCl buffer (pH 8.5) containing 4, 2, 1, 0.5, or 0 M urea. Apparent molecular masses of rFTXs of precursor and mature forms were estimated to be 33 or 31 kDa, respectively, on SDS-PAGE (Fig. 7B) and Western immunoblotting using anti-FTX serum (results not shown). The N-terminal sequences of both rFTXs were identical with that of FTX (results not shown), suggesting that the initial Met was cleaved in the E. coli cells. Molecular masses (M ϩ H) ϩ of the precursor and mature forms were estimated to be 29,966 and 27,854 Da, respectively, by MALDI-TOF MS ( Fig.  9C and results not shown). These values coincided well with the calculated masses (M ϩ H) ϩ , 29,965 and 27,816 Da, for the FTX molecules with or without the C-terminal 20 residues, respectively. Thus, rFTXs of the precursor and mature forms consisted of 271 or 251 residues and were designated rFTX271 and rFTX251, respectively. Molecular sizes of native rFTX271 and rFTX251 were analyzed by GPC using a TSKgel G3000SW column. As shown in Fig. 7C, rFTX271 and rFTX251 were eluted at the positions corresponding to 62 or 35 kDa, respectively, suggesting that rFTX271 was a dimer in solution, whereas rFTX251, like natural FTX (14), existed as a monomer in solution. To confirm this conclusion, a chemical cross-linking experiment was performed using a lower concentration of rFTX (150 nM); rFTX271 (5.0 g/ml) or rFTX251 (4.6 g/ml) was treated with 0.05% (w/v) glutaraldehyde, followed by Western immunoblotting. As shown in Fig. 7D, a band corresponding to 66 kDa was formed upon the treatment of rFTX271 with glutaraldehyde, whereas no band corresponding to dimers was visible with rFTX251 under the same conditions.
Maturation of Dimeric Precursor to Monomeric Active Form by C-terminal Truncation-A hemolytic assay indicated that rFTX251 lysed human erythrocytes in a dose-dependent manner at concentrations of 0.2-2.0 g/ml, and the hemolytic activity of rFTX251 was comparable with that of natural FTX (Fig. 8A). In contrast, rFTX271 exhibited no significant activity at high concentrations of up to 100 g/ml (Fig. 8A). Oligmer formation by rFTX was tested by the experiments including solubilization of rFTX-treated human erythrocytes with 2% SDS at 25°C and Western immunoblotting. As shown in Fig.  8B, two immunostained bands corresponding to 31 or 180 kDa were visible when human erythrocytes were incubated with rFTX251. In contrast, no band corresponding to a high molecular mass complex of rFTX271 was visible, although two immunostained bands corresponding to 33 or 66 kDa were detected with the rFTX271-treated cells (Fig. 8B). The results indicated that rFTX251, like natural FTX (14), assembled into a SDS-stable, 180-kDa complex on human erythrocytes, whereas dimeric rFTX271 bound to the cells as efficiently as rFTX251 but failed to form an oligomer there. The membranes of the rFTX251-treated erythrocytes were solubilized with 2% SDS and fractionated by a sucrose density gradient ultracentrifugation, and the fractions obtained were subjected to Western immoblotting using anti-FTX serum. As a result, the immunostained band corresponding to 180 kDa was detected in the fractions of 20 -25% (w/w) sucrose (results not shown), as shown in the previous study (14). Electron microscopy for the fractions indicated the presence of the ring-shaped structures with outer and inner diameters of 10 and 5 nm, respectively (Fig. 8C). Thus, monomeric rFTX251, like natural FTX (14), bound to and assembled into a ring-shaped pore complex on human erythrocytes, whereas dimeric rFTX271 bound to the cells but failed to form a pore complex.
To study the maturation process of FTX, rFTX271 was treated with TPCK-treated trypsin at 37°C for 0 -24 h at an enzyme/substrate weight ratio of 1:10, followed by SDS-PAGE and hemolytic assay for the trypsin-treated protein. As shown in Fig. 9A, rFTX271 was cleaved by trypsin to form the band corresponding to 31 kDa, and thereafter the 29-kDa molecules were generated by the prolonged incubation. Hemolytic activity of the reaction mixture increased in tandem with the conversion of rFTX271 (33 kDa) to the 31-and 29-kDa molecules (Fig.  9, A and B). To determine the cleavage site(s), rFTX271 was cleaved with TPCK-treated trypsin at 37°C for 4 or 24 h in an enzyme/substrate weight ratio of 1:70, and the reaction mixtures were subjected to MALDI-TOF MS. Without the trypsin digestion, molecular mass ((M ϩ H) ϩ ) of rFTX271 was determined to be 29,972.3 Da, which coincided well with the calculated value (29,964.2 Da) of the protein (Fig. 9C, lower panel).  (Fig. 9C, upper  panel). Furthermore, the molecular size of the trypsin-treated rFTX271 was estimated by GPC using a TSKgel G3000SW FIG. 7. Expression of rFTX271 and rFTX251, and their dimeric and monomeric states in solution. A, constructs of pFTX272 and pFTX252, the expression vectors for production of rFTX271 and rFTX251, respectively. Amino acids are denoted with one-letter codes and numbered as in Fig. 1. B, SDS-PAGE for purified rFTX271 and rFTX251. The gels were stained with Coomassie Brilliant Blue R-250. C, GPC of rFTX271, rFTX251, and trypsin-treated rFTX271 on a TSKgel G3000SW column. Elution was conducted with 10 mM sodium phosphate buffer, pH 6.8, containing 0.5 M NaCl at a flow rate of 0.5 ml/min. rFTX271 was digested with TPCK-treated trypsin at 37°C for 24 h at an enzyme/substrate weight ratio of 1:70. Bovine serum albumin, ovalbumin, and chymotrypsinogen A were used as molecular mass standards. D, cross-linking of rFTX. rFTX271 (5.0 g/ml) or rFTX251 (4.6 g/ml) was treated with or without 0.05% (w/v) glutaraldehyde at 25°C for 20 min, followed by Western immunoblotting using anti-FTX serum. GA, glutaraldehyde.
column. As shown in Fig. 7C (trypsin-treated rFTX271), the trypsin-treated rFTX271 gave a large peak corresponding to 35 kDa as well as a small peak corresponding to 62 kDa. The trypsin-treated rFTX271 also exhibited hemolytic activity at a level of 5000 units/mg protein (results not shown). Thus, dimeric rFTX271 was converted to hemolytically active monomers by cleavage of the linkage between Lys 251 and Met 252 . DISCUSSION In this study, we established the complete amino acid sequence of FTX from the sequence information obtained by Edman degradation of intact PE-FTX and the peptides arising from PE-FTX by several enzymatic and chemical cleavages, together with the compositional analyses and MALDI-TOF MS for the peptides. As a result, FTX consisted of 251 residues, indicating that the cloned cDNA encoded a FTX precursor with the initial Met and an additional C-terminal 20 residues. Analyses of the molecular and pore-forming properties of rFTX showed that dimeric FTX precursor was converted by the cleavage at the linkage between Lys 251 and Met 252 to monomers, which bound to and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes. Activation of protoxin by a C-terminal truncation has been reported to several poreforming cytolysins including aerolysin from A. hydrophila (26,27), Clostridium septicum ␣-toxin (28), enterobin from the Brazilian plant Enterolobium contortisiliquum (29), and Pseudomonas aeruginosa cytotoxin (30), and all of the precursors were shown to be dimers in solution. Previous studies showed that aerolysin, C. septicum ␣-toxin, and enterobin remain dimers after the C-terminal truncation, and the processed dimers bind to and assemble into pore complexes on the target cells (26 -29). In contrast, the precursors of FTX and P. aeruginosa cytotoxin are converted to active monomers by the cleavage of the Cterminal 20 residues (this study) (30). Thus, the C-terminal segment of FTX precursor is involved in the formation of stable dimer. According to an analysis of the hydropathy profile using the algorithm of Kyte and Doolittle (31) and a secondary structure prediction using the algorithm of Garnier et al. (32), the 7-residue segment containing the cleavage site (i.e. Lys 251 -Met 252 ) forms a hydrophilic ␣-helix (Fig. 10A), which disappears by the removal of the C-terminal 20 residues (Fig. 10B). Because the linkage between Lys 251 and Met 252 is highly sus- ceptible to trypsin (Fig. 9C), the C-terminal part of the FTX precursor would be a surface-exposed, flexible segment, interacting with the C-terminal segment of another precursor molecule to form a stable dimer. Alternatively, like the Cterminal segment of proaerolysin, the C-terminal ␣-helix of the FTX precursor may be required for stabilization of a certain conformation of the protein in a dimer. Based on the x-ray crystallographic analysis (33), the C-terminal region of proaerolysin would mask hydrophobic patches of the toxin molecule to inhibit oligomerization of the toxin on the membranes. Further study is needed to elucidate how the Cterminal segment of FTX precursor contributes to dimer formation.
Protein sequencing of FTX by Edman degradation was somewhat hampered by the difficulty in the analysis of the central part of the molecule. The sticky nature of this region prevented recovery of the peptides of this region from enzyme digests. Relatively large peptides of this region were recovered only in denaturing solvents, 6 M guanidine hydrochloride or strongly acidic solutions. Thus, multiple procedures for chemical cleavages including the hydrolysis of T7k peptide with 12 M HCl were needed for the sequence analysis of the central part. A combination of a hydropathy analysis and a secondary structure prediction revealed that FTX is a rather hydrophilic protein with a relatively hydrophobic central part, where hydrophobic ␤-sheets may interact with each other to form a rigid structure (Fig. 10). Upon contact with membrane, FTX forms an oligomeric pore structure (14). The central part of FTX may be responsible for the oligomerization and/or the membrane insertion of the pore structure. In addition to the difficulty in the analysis of the central part, the heterogeneity in the C terminus of FTX delayed completion of the sequence analysis. We concluded that FTX consists of 251 residues on the basis of the fact that no peptide extending from Lys 251 was detected among the peptides arising from PE-FTX by the cleavage with CNBr (Fig. 2, A-C, and Table I), Achromobacter protease I (Fig.  2D and Table II), or S. aureus V8 protease (Fig. 3, A and B, and results not shown). The conclusion is consistent with the finding that rFTX271 was cleaved by trypsin preferentially at the linkage between Lys 251 and Met 252 (Fig. 9C), irrespective of the presence of two more Lys residues in the C-terminal segment (i.e. Lys 254 and Lys 260 ; Fig. 6). The resultant 251-residue protein, FTX, may be thereafter cleaved by carboxypeptidase(s) to generate heterogenous C termini in the mushroom and/or in the purification steps. Furthermore, some FTX preparations exhibited another band corresponding to 29 kDa on SDS-PAGE/Western immunoblotting, and cleavage at the linkages between Lys 241 and Thr 242 or between Lys 240 and Lys 241 was indicated by MALDI-TOF MS (Fig. 9C). The amount of the 29-kDa molecules increased during storage at 4°C in the absence of protease inhibitors, 2 producing more heterogeneity in the C terminus. Incidentally, the presence of the C-terminally truncated molecular species may interpret, at least in part, the higher hemolytic activity of natural FTX, compared with that of rFTX251 (i.e. FTX and rFTX251 caused 50% hemolysis at the protein concentration of 0.5 or 0.7 g/ml, respectively) ( Fig.  8A), because C-terminally truncated FTX with an apparent mass of 29 kDa lysed human erythrocytes 3-fold more efficiently than intact FTX.