A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn2+-depleted ProB can regain the activity with Zn2+, Co2+, or Mn2+. Specific cleavage of Pro29-LYS30 in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn2+ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.
Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.
The complete amino acid sequences of two lysine-specific zinc metalloendopeptidases (EC 3.4.24), Grifola frondosa metalloendopeptidase (GFMEP) and Pleurotus ostreatus metalloendopeptidase (POMEP), from the fruiting bodies of these two edible mushrooms have been established based on the sequence information of the peptides generated from the reduced and alkylated GFMEP and POMEP by proteolytic digestions using GFMEP, trypsin, and other proteinases as well as by several chemical cleavages. From the sequences, it was found that GFMEP and POMEP were polypeptides composed of 167 and 168 amino acid residues, from which their molecular weights were calculated to be 18,040.5 and 17,921.3 in accord with the observed (M+H)+ values of 18,028 and 17,927, respectively, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two disulfide bonds in GFMEP were found to link Cys5 to Cys75 and Cys77 to Cys97. An unusual post-translational modification of GFMEP was corroborated to be a partial attachment of a single mannose to Thr42. Comparison of the sequences revealed that overall identity between the enzymes was 61.3%. Although a highly homologous sequence was not found in sequence data bases except for a consensus zinc-binding sequence, HEXXH, both metalloendopeptidases somewhat resembled a family of metalloproteinases categorized as deuterolysin. These proteases together with GFMEP and POMEP do not have conserved third and/or fourth liganding amino acid residues seen in metzincin or thermolysin superfamily proteins and belong to a novel zinc metalloendopeptidase superfamily.
A number of peptides with different lengths corresponding to various regions of human pulmonary surfactant protein SP-C were synthesized and their activity evaluated to improve in vitro surface activities and in situ lung pressure-volume characteristics of a ternary lipid mixture composed of dipalmitoylphosphatidylcholine, phosphatidylglycerol and palmitic acid (75:25:10, w/w). SP-C (1-35), a synthetic peptide with the entire length of human SP-C, and some other peptides with various lengths of its partial sequences were remarkably active. All of these peptides shared a common core sequence of (C)CPVHLKRLLIVVVVVVLIVVVIVGAL(L). Any deletion in this core sequence resulted in reduction of activity of the peptide. SP-C (5-31) and SP-C (6-32), the minimum peptides containing the core sequence, were combined with the ternary lipid mixture at the final peptide concentration of 2% (w/w) into synthetic surfactants which showed excellent properties comparable with those of Surfacten, a commercially available modified bovine lung surfactant. In a Langmuir-Wilhelmy surface balance, the synthetic surfactant containing SP-C (6-32) spread and adsorbed quickly to reach a surface tension of 30.8 mN/m at 30-s spreading time and 41.2 mN/m at 1-min adsorption time, respectively; the presence of SP-C (6-32) significantly prevented the decrease of surface activity of the ternary lipid mixture during dynamic compression-expansion cycles. Furthermore, tracheal instillation of the synthetic surfactant containing SP-C (6-32) at the dose of 50 mg of phospholipids/kg improved lung pressure-volume characteristics of immature rabbit neonates to a level similar to that of mature neonates at term.
Journal Article Purification and Characterization of Acid Phosphodiesterases of Cultured Tobacco Cells Get access Hiroshi Matsuzaki, Hiroshi Matsuzaki Department of Biochemistry, Faculty of Science, The University of Saitama, Urawa, Saitama 338, Japan Search for other works by this author on: Oxford Academic Google Scholar Yohichi Hashimoto Yohichi Hashimoto Department of Biochemistry, Faculty of Science, The University of Saitama, Urawa, Saitama 338, Japan Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 45, Issue 6, 1 June 1981, Pages 1317–1325, https://doi.org/10.1080/00021369.1981.10864711 Published: 01 June 1981 Article history Received: 17 September 1980 Published: 01 June 1981
The partial structure of a spore germination inhibitor from a cellular slime mold, Dictyostelium descoideum was investigated. The molecular weight of this substance is 412 daltons. It contains at least N-(3-methyl-2-butenyl) adenine and an unknown α-amino acid residue. At the concentration of _??_0.03 μg/ml (_??_10-8M), this compound completely inhibited the spore germination of this organism.
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 fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 m guanidine HCl. The 7-kDa peptide was cleaved with 12 m HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys251 and Met252 by trypsin. 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 fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 m guanidine HCl. The 7-kDa peptide was cleaved with 12 m HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys251 and Met252 by trypsin. Pore-forming cytolytic proteins are distributed in a wide variety of eukaryotic and prokaryotic organisms (1Bernheimer A.W. Rudy B. Biochim. Biophys. Acta. 1986; 864: 123-141Crossref PubMed Scopus (250) Google Scholar, 2Bhakdi S. Tranum-Jensen J. Rev. Physiol. Biochem. Pharmacol. 1987; 107: 147-223Crossref PubMed Google Scholar). Complement, perforin from the cytotoxic T-cells, α-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 (2Bhakdi S. Tranum-Jensen J. Rev. Physiol. Biochem. Pharmacol. 1987; 107: 147-223Crossref PubMed Google Scholar, 3Esser A.F. Toxicol. 1997; 87: 229-247Crossref Scopus (105) Google Scholar, 4Menstrina G. Dalla Serra M. Comai M. Coraiola M. Viero G. Werner S. Colin D.A. Monteil H. Prevost G. FEBS Lett. 2003; 552: 54-60Crossref PubMed Scopus (116) Google Scholar, 5Montoya M. Gouaux E. Biochim. Biophys. Acta. 2003; 1609: 19-27Crossref PubMed Scopus (95) Google Scholar, 6Parker M.W. Toxicon. 2003; 42: 1-6Crossref PubMed Scopus (17) Google Scholar). The self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly, membrane insertion, and molecular architecture of transmembrane pores (3Esser A.F. Toxicol. 1997; 87: 229-247Crossref Scopus (105) Google Scholar, 4Menstrina G. Dalla Serra M. Comai M. Coraiola M. Viero G. Werner S. Colin D.A. Monteil H. Prevost G. FEBS Lett. 2003; 552: 54-60Crossref PubMed Scopus (116) Google Scholar, 5Montoya M. Gouaux E. Biochim. Biophys. Acta. 2003; 1609: 19-27Crossref PubMed Scopus (95) Google Scholar, 6Parker M.W. Toxicon. 2003; 42: 1-6Crossref PubMed Scopus (17) Google Scholar). 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 (7Faulstich H. Buehring H.J. Seitz J. Biochemistry. 1983; 22: 4574-4580Crossref PubMed Scopus (16) Google Scholar, 8Wilmsen H.U. Faulstich H. Eibl H. Boheim G. Eur. Biophys. J. 1985; 12: 199-209Crossref PubMed Scopus (13) Google Scholar, 9Lin J.Y. Lin Y.J. Chen C.C. Wu H.L. Shi G.Y. Jeng T.W. Nature. 1974; 252: 235-237Crossref PubMed Scopus (37) Google Scholar, 10Tomita T. Noguchi K. Mimuro H. Ukaji F. Ito K. Sugawara-Tomita N. Hashimoto Y. J. Biol. Chem. 2004; 279: 26975-26982Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). 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 (11Fernandez Espinar M. Labarere J. Curr. Genet. 1997; 32: 420-424Crossref PubMed Scopus (49) Google Scholar). 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 (12Berne S. Krizaj I. Pohleven F. Turk T. Macek P. Sepcic K. Biochim. Biophys. Acta. 2002; 1570: 153-159Crossref PubMed Scopus (87) Google Scholar). Lin et al. (9Lin J.Y. Lin Y.J. Chen C.C. Wu H.L. Shi G.Y. Jeng T.W. Nature. 1974; 252: 235-237Crossref PubMed Scopus (37) Google Scholar) isolated a cardiotoxic and cytolytic 22-kDa protein from the basidiocarps of the edible mushroom Flammulina velutipes, and designated it flammutoxin (FTX). 1The abbreviations used are: FTX, flammutoxin; rFTX, recombinant FTX; rFTX271, recombinant FTX consisting of 271 residues; rFTX251, recombinant FTX consisting of 251 residues; TPCK, N-tosyl-L-phenylalanyl-chloromethylketone; PE, S-pyridylethylated; HPLC, high performance liquid chromatography; RP-HPLC, reversed phase HPLC; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry. Later, Bernheimer and Oppenheim (13Bernheimer A.W. Oppenheim J.D. Toxicon. 1987; 25: 1145-1152Crossref PubMed Scopus (17) Google Scholar) 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. (9Lin J.Y. Lin Y.J. Chen C.C. Wu H.L. Shi G.Y. Jeng T.W. Nature. 1974; 252: 235-237Crossref PubMed Scopus (37) Google Scholar) derived from their 32-kDa FTX by partial proteolysis. We isolated FTX as a 31-kDa single hemolysin of F. velutipes, determined the N-terminal 28 residues, and studied the molecular basis of the cytolytic action of the protein (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). 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 (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). By using planar lipid bilayers, we showed that FTX forms a cation-selective, voltage-gated channel with a diameter of 4–5 nm (15Tadjibaeva G. Sabirov R. Tomita T. Biochim. Biophys. Acta. 2000; 1467: 431-443Crossref PubMed Scopus (21) Google Scholar). Watanabe et al. (16Watanabe H. Narai A. Shimizu M. Eur. J. Biochem. 1999; 262: 850-857Crossref PubMed Scopus (27) Google Scholar) 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 (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar, 16Watanabe H. Narai A. Shimizu M. Eur. J. Biochem. 1999; 262: 850-857Crossref PubMed Scopus (27) Google Scholar). Watanabe et al. (16Watanabe H. Narai A. Shimizu M. Eur. J. Biochem. 1999; 262: 850-857Crossref PubMed Scopus (27) Google Scholar) 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 (16Watanabe H. Narai A. Shimizu M. Eur. J. Biochem. 1999; 262: 850-857Crossref PubMed Scopus (27) Google Scholar). However, they did not express the cloned cDNA. Concurrently with their cloning, we cloned a cDNA encoding the same protein (GenBank™ 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. (17Sakamoto Y. Azuma T. Ando A. Tamai Y. Miura K. Mycoscience. 2000; 41: 279-282Crossref Scopus (1) Google Scholar) studied expression of genes in different developmental stages of F. velutipes and cloned C1 cDNA (GenBank™ accession number AB030006), which was specifically expressed during the fruiting body formation. A search on the DDBJ/GenBank™/EBI nucleotide sequence data bases indicated that the C1 cDNA is identical with the cDNAs cloned by us and by Watanabe et al. (16Watanabe H. Narai A. Shimizu M. Eur. J. Biochem. 1999; 262: 850-857Crossref PubMed Scopus (27) Google Scholar). The same group also analyzed chronological expression of proteins in F. velutipes by using two-dimensional 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 (17Sakamoto Y. Azuma T. Ando A. Tamai Y. Miura K. Mycoscience. 2000; 41: 279-282Crossref Scopus (1) Google Scholar). 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™ 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. Materials—Basidiocarps of F. velutipes were purchased from the producers in Gunma and Miyagi Prefecture and stored at –40 °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 (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). FTX was reduced and pyridylethylated (PE) as described (18Nonaka T. Dohmae N. Hashimoto Y. Takio K. J. Biol. Chem. 1997; 272: 30032-30039Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Chemical Cleavages—Methionyl bonds of PE-FTX were cleaved according to the method of Gross (19Gross E. Methods Enzymol. 1967; 11: 238-255Crossref Scopus (765) Google Scholar) 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 (20Bornstein P. Balian G. Methods Enzymol. 1977; 47: 132-145Crossref PubMed Scopus (320) Google Scholar). 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 (21Landon M. Methods Enzymol. 1977; 47: 145-149Crossref PubMed Scopus (257) Google Scholar). T7k peptide was hydrolyzed in 12 m HCl at room temperature for 15 h, as described by Titani and Narita (22Titani K. Narita K. J. Biochem. (Tokyo). 1964; 56: 241-256Crossref PubMed Scopus (11) Google Scholar). 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 TPCK-treated trypsin at 37 °C for 18 h in 50 mm Tris-HCl buffer (pH 8.3) containing 4 m urea and 10 mm CaCl2 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 CaCl2, 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 G2000SWXL and TSKgel G3000SWXL (7.8 × 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 × 100 or 4.6 × 100 mm; Applied Biosystems, Foster City, CA), an Aquapore PH-300 (2.1 × 30 mm; Applied Biosystems), a TSKgel super ODS column (2.0 × 48 mm; Tosoh), or a Nova-Pak C18 column (3.9 × 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 (23Bidlingmeyer B.A. Cohen S.A. Tarvin T.L. J. Chromatogr. 1984; 336: 93-104Crossref PubMed Scopus (2149) Google Scholar). 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 α-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. 5′-ATGGATCCICARGTIAARACITCITGGGARGAYYT-3′, 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 (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). The antisense primer was 5′-GCAAGCTTTTTTTTTTTTTTTTTTTTTTTT-3′, 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 ∼1 kbp were inserted into the SmaI site of pUC118 and sequenced. The resultant plasmid was designated pUF1. Amplification of the 5′-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 3′-ends of the reverse transcripts by using terminal deoxyribonucleotidyl transferase (TaKaRa). The 5′-end of the FTX cDNA was amplified by a PCR using the oligo(dT) primer and an antisense primer (i.e. 5′-CGCTCAATGGAAACTATCTCACGA-3′, which corresponds to the segment from the 469th to the 498th nucleotide of the FTX cDNA; Fig. 7). Amplified DNA fragments of ∼600 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 5′-CCTCAAGTCAAGACAAGTTGGGAGGATCTC-3′, which corresponds to the N-terminal 10 amino acid residues of FTX (Fig. 7), and the antisense primer was 5′-GGTGTCGACTCCGTAGAAATCGAAATCTCG-3′, 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 ∼400 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 5′-ATTCTGCAGTTGAGTCAGTCGATCACC-3′ (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 5′-CTCAAGCTTTCACTTCACCGTCAAAGGGGCAG-3′ (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 ∼400 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™/EBI nucleotide sequence databases. Expression, Renaturation, and Purification of rFTX—E. coli DH5α cells harboring pFTX272 or pFTX252 were grown at 37 °C in 2× YT medium (1.6% Bacto-Trypton, 1% Bacto-Yeast extract, and 0.5% NaCl; Difco) with ampicillin (100 μg/ml). When optical density at 660 nm of the culture reached 0.4, isopropyl-β-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/cm2 and was centrifuged at 14,000 × 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 × 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 × 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 × 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 (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (220342) Google Scholar). Hemolytic Assay—Hemolytic assay was performed as described (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). Human erythrocytes (3 × 107 cells/ml) were incubated with rFTX (the final concentrations 0.1–100 μg/ml) at 25 °C for 30 min. After centrifugation at 600 × 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 hemolytic 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 μg/ml, respectively) was treated with 0.05% (w/v) glutaraldehyde at 20 °C for 20 min as described (25Sugawara-Tomita N. Tomita T. Kamio Y. J. Bacteriol. 2002; 184: 4747-4756Crossref PubMed Scopus (47) Google Scholar). 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 (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). Assembly of rFTX into Membrane Pore Complex—Complex formation by rFTX was assayed as described (14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). rFTX (0.5–2.0 μg) was incubated with human erythrocytes (1.0 × 108 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 (10Tomita T. Noguchi K. Mimuro H. Ukaji F. Ito K. Sugawara-Tomita N. Hashimoto Y. J. Biol. Chem. 2004; 279: 26975-26982Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Tomita T. Ishikawa D. Noguchi T. Katayama E. Hashimoto Y. Biochem. J. 1998; 333: 129-137Crossref PubMed Scopus (26) Google Scholar). Human erythrocytes (3 × 109 cells) were incubated with rFTX271 or rFTX251 (50 μg 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 μg/ml) was treated with TPCK-treated trypsin (10 μg/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 μg/ml) and were subjected to SDS-PAGE or hemolytic assay. For MALDI-TOF MS, rFTX271 (28 μg/ml) was treated with TPCK-treated trypsin (0.4 μg/ml) at 37 °C for 4 or 24 h, and small portions were immediately withdrawn and analyzed as described above. 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. ∼70% 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 G2000SWXL (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
