968 Regular Article Biol. Pharm. Bull. 37(6) 968–978 (2014) Vol. 37, No. 6

X-Ray Crystallographic Structure of RNase Po1 That Exhibits Anti- tumor Activity Hiroko Kobayashi,*,a Takuya Katsutani,b Yumiko Hara,b Naomi Motoyoshi,a Tadashi Itagaki,a Fusamichi Akita,b Akifumi Higashiura,b Yusuke Yamada,c Norio Inokuchi,a and Mamoru Suzuki*,b a School of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan: b Institute for Protein Research, Osaka University; 3–2 Yamadaoka, Suita, Osaka 565–0871, Japan: and c Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization KEK; 1–1 Oho, Tsukuba, Ibaraki 305–0801, Japan. Received November 29, 2013; accepted March 12, 2014

RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus os- treatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disul- fide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic po- tential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein. Key words ribonuclease; crystal structure; anti-tumor activity; Pleurotus ostreatus

RNase Po1 hydrolyzes single-stranded RNA via a 2′,3′-cy- not RNase Po1 has three disulfide bonds, and which cysteine clic phosphate intermediate at the 3′-terminus of oligonucle- residues may form those bonds. Furthermore, RNase Po1 is otides, and is a guanylic acid-specific ribonuclease (RNase) (a an alkaline protein (isoelectric point (pI) 9.0), whereas RNase member of the RNase T1 family of RNases). RNase Po1 has a T1 (pI 2.9) and most members of the RNase T1 family of molecular mass of approximately 11 kDa and exhibits optimal RNases are acidic proteins (pI 4.0–4.5).24,25) Thus, RNase Po1 activity at pH 7.5, similar to RNase T1 from Aspergillus ory- is a unique member of the RNase T1 family. Recently, we zae, the best-known member of this family.1,2) We previously reported that RNase Po1 exhibits anti-tumor activity towards isolated and purified RNase Po1 from Pleurotus ostreatus several types of human tumor cells,26) in contrast to RNase of the basidiomycota and reported its amino acid sequence.1) T1 and other RNase T1 family RNases that are non-toxic to There is high sequence identity (40%) between RNase Po1 and tumor cells, except for α-sarcin from Aspergillus giganteus.27) RNase T1. The X-ray crystallographic structures of RNase α-Sarcin is a single polypeptide chain protein composed of T13) and RNase Ms4) have been reported, and the catalytic site 150 amino acids, bigger than RNase Po1 (101 amino acids) and base recognition region for RNase activity have been elu- and with low sequence identity.28) α-Sarcin exhibits anti-tumor cidated. The catalytic site of RNase T1 consists of His40 and/ activity by degrading the larger ribosomal RNA of tumor or Glu58, Arg77, and His92.5) Moreover, Steyaert et al.6) and cells.29) The three-dimensional structure of α-sarcin has been Nonaka et al.7) reported that Glu58, rather than His40, must solved,30) and it is necessary to compare its structure with be the general base catalyst of the RNase T1 family. The base that of RNase Po1 to understand the basis of their anti-tumor recognition site of RNase T1 consists of Tyr42, Asn43, Asn44, activities. Tyr45, Glu46, and Asn98.5) These residues are completely There is another RNase family whose members have conserved in RNase Po1 except for Tyr45 (RNase T1) being molecular masses of 13–14 kDa. This family is pyrimidine changed to Phe (RNase Po1). A comparison of the primary base-specific and is referred to as the RNase A family. Al- structures of RNase Po1 and RNase T1 and other RNase T1 most all RNase A family members are non-toxic, but some family RNases shows that RNase T1 contains four cysteine RNases have been reported to exhibit anti-tumor activity.31–35) residues that form two disulfide bonds, whereas RNase Po1 RNases from Rana pipiens are the most extensively studied, has six cysteine residues1,8–23) (Fig. 1). The C9–C99 disulfide and ranpirnase (Onconase) is currently in clinical trials as an bond of RNase Po1 is superimposable on the analogous di- anti-tumor drug.36,37) The X-ray crystallographic structures of sulfide bond in RNase T1 (C6–C103). This disulfide bond is RNase A and Onconase have been reported.38,39) Comparisons conserved in all known RNase T1 family enzymes, except for of their structures suggest a relationship between stability and those of bacterial origin. The C48–C82 bond of RNase Po1 anti-tumor activity.40,41) In the present work, we determined is superimposable on RNase U1 and RNase U2 from Usti- the X-ray crystallographic structure of RNase Po1 and in- lago sphaerogena. Therefore, these two disulfide bonds may vestigated the relationship between structure and anti-tumor exist in RNase Po1. The other cysteine residues in RNase Po1 activity by comparing the X-ray structures of RNase Po1 and (Cys7, Cys84) are not found in RNase T1 and other RNase T1 RNase T1, which have high sequence identity. family RNases. Therefore, we have not considered whether or

The authors declare no conflict of interest.

* To whom correspondence should be addressed. e-mail: [email protected]; © 2014 The Pharmaceutical Society of Japan [email protected] June 2014 969

Fig. 1. Comparison of the Amino Acid Sequences of RNase Po1 or RNase T1 with Those of RNases Belonging to the RNase T1 Family Po1: Pleurotus ostreatous RNase,1) U1, U2: Ustilago sphaerogena RNase,8,9) F1: Fusarium moniliforme RNase,10) FL1: Fusarium lateritium RNase,11) Th1: Trichoderma harzuanum RNase,12) Ms: Aspergillus saitoi RNase,13) T1: Aspergillus oryzae RNase,14) C2: Aspergillus clavatus RNase,15) Ap1: Aspergillus pallidus RNase,16) N1: Neu- rospora crassa RNase,17) Pch1: Penicillium crysogenum RNase,18) Pb1: Penicillium brevicompactum RNase,19) Sa: Streptomyces aureofaciens RNase,20) St: Streptomyces rythreus RNase,21) Bi: Bacillus intermedius RNase,22) Ba: Bacillis amyloliquefacience RNase.23) * Catalytic site. # Base recognition site. Disulfide bonds of RNases are shown as connected by bold lines.

MATERIALS AND METHODS Table 1. Data Collection and Refinement Statistics

Enzymes RNase Po1 was expressed in Escherichia coli. Parameter RNase Po1 The cDNA was ligated to expression vector pET-pel-Po1, Data collection constructed following the procedure of Huang et al.42) from Beamline Photon Factory BL-17A pET22b (Novagene, Darmstadt, Germany), then transferred to Wavelength (Å) 0.9800

E. coli BL21(DE3) pLysS (Novagene). The cells were cultured Space group P31 in Terrific Broth at 25°C for 7 d, with the addition of 100 µg/ Cell dimension (Å) a=b=75.56, c=34.80 mL of ampicillin and a final concentration of 0.5 mM isopropyl Resolution (Å) 37.78–1.85 (1.95–1.85) β-D-1-thiogalactopyranoside (IPTG) (Wako Pure Chemical Reflections measured 136098 Industries, Ltd., Osaka, Japan). The supernatant of the culture Unique reflections 18553 was used for subsequent purification steps. The supernatant Redundancy 7.7 (5.3) was fractionated with 90% saturated ammonium sulfate, and Completeness (%) 99.6 (84.7) a) the precipitate was collected by centrifugation at 10000 rpm Rmerge (%) 15.7 (62.7) Mean I/σ(I) 10.1 (2.5) for 30 min. The precipitate was suspended in 10 mM acetate buffer (pH 6.0) and dialyzed overnight against de-ionized Refinement Resolution (Å) 32.72–1.85 water. The dialysate was heated at 60°C for 10 min and then Number of reflections 18541 rapidly cooled in ice-cold water for 10 min. The precipitate R b)/R c) 0.164/0.176 was recovered by centrifugation at 10000 rpm for 30 min. Sub- work free R.m.s deviations sequent purification steps were carried out using previously 26) Bond length (Å) 0.0034 described protocols. Bond angles (°) 0.782 Enzyme Assay RNase activity was measured as de- Ramachandran analysis scribed previously using yeast RNA (Marine Biochemicals, Favored/allowed (%) 98.7/1.3 Tokyo, Japan) as the substrate at pH 7.5 at 37°C.43) Values in parentheses are for the highest-resolution shell. a) Rmerge=∑hkl ∑i |I(hkl; i)− Protein Concentration The protein concentration of the 〈I(hkl)〉|/∑hkl ∑i I(hkl; i), where I(hkl; i) is the intensity of an individual mea- final enzyme preparation was determined spectrophotometri- surement, and 〈I(hkl)〉 is the average intensity from multiple observations. b) R =S||F |−|F ||/|F |, where |F | and |F | are the observed and calculated cally, assuming an absorbance of 0.54 for a 0.1% solution at work obs calc obs obs calc structure factor amplitudes, respectively. c) Rfree is the same as Rwork, but for a 10.0% 280 nm. This value was estimated from the amino acid com- subset of all reflections for RNase Po1. position of RNase Po1 (data not shown). Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel (3.5 M sodium formate, 0.1 M 1,3-bis[tris(hydroxymethyl)- Electrophoresis (Tricine-SDS-PAGE) Tricine-SDS-PAGE methylamino] propane-1,3-diol (Bis-Tris propane) (pH 7.0) in was performed using a 15% polyacrylamide gel by Schagger’s SaltRx1 (Hampton Research, Aliso Viejo, CA, U.S.A.). Drops method.44) Proteins on the gel were stained by silver stain- were placed over a well containing 500 µL of reservoir solu- ing. Activity staining of the RNases was conducted using the tion (4 M sodium formate, 0.1 M Bis-Tris propane, pH 7.0, 10% method of Blank et al.45) polyethylene glycol (PEG) 400). Crystals suitable for data col- Crystallization of RNase Po1 RNase Po1 was crys- lection were obtained in drops containing 0.5 µL protein solu- tallized at 293 K by the hanging drop vapor diffusion tion (10 mg/mL RNase Po1 in 20 mM Tris–HCl, pH 7.5) mixed method. An initial crystal was obtained using condition #31 with 0.5 µL reservoir solution (4 M sodium formate, 0.1 M Bis- 970 Vol. 37, No. 6

Tris propane, pH 7.0, 10% PEG 400) and 0.3 µL 2 M cesium chloride after 3 d. Data Collection and Determination of the Structure Diffraction data were measured at 100 K and a wavelength of 0.9800 Å using an ADSC Quantum 270 detector on beamline BL-17A at the Photon Factory (Tsukuba, Japan). Data from a single crystal were integrated and scaled using XDS46) and SCALA.47) The crystal belonged to trigonal space group

P31. The structure was determined by molecular replacement with BALBES48) and MOLREP.49) The presence of the three subunits of RNase Po1 in an asymmetric unit gave a crystal volume per protein mass (VM) of 1.78 Å3 Da−1 and a corre- sponding solvent content of 31.0%.50) Structure refinement was 51) performed using PHENIX. The molecular model was manu- Fig. 2. Tricine-SDS-PAGE (15% Slab-Gel) of RNase Po1 52) ally corrected in the electron density map using COOT. The Purified RNase Po1 was homogeneous according to Tricine-SDS-PAGE. Tricine- SDS-PAGE was performed using a 15% polyacrylamide gel. (a) Silver staining of final Rwork and Rfree values were 0.164 and 0.176, respectively. molecular marker proteins. (b) Silver staining of RNase Po1. (c) Activity staining of The Ramachandran statistics for the structure calculated using RNase Po1 using RNA as the substrate.

Fig. 3. Tertiary Structures of RNase Po1 and RNase T1 The figure was drawn with PyMOL (http://pymol.sourceforge.net). (a), (b) The N- and C-termini are labeled N and C, respectively. The α-helices and β-strands are marked α1 and β1–7, respectively. The circles enclose disulfide bonds. (a) RNase T1 is colored pink (PDB ID: 2BU4, Proteins 1999, 36, 117–134), 2′GMP is shown as sticks colored blue and red. (b) RNase Po1 is colored blue (PDB ID: 3WHO). (c) Structural overlay of RNase Po1 with that of the RNase T1/2′GMP complex. Active site residues of RNase Po1 and RNase T1 are colored blue and pink, respectively. The α-helices and β-strands are marked α1 and β1–7, respectively. The disulfide bonds of RNase Po1 are shown as sticks colored yellow. (d) The active site of RNase Po1 superimposed with that of the RNase T1/2′GMP complex from (c). 2′GMP is gray. In (c) and (d), the amino acid numbers of RNase Po1 are shown first and those of RNase T1 follow in parentheses. June 2014 971

MolProbity53) were as follows: favored regions, 98.7%; allowed buffered saline (PBS) as a control in RPMI 1640 medium regions, 1.3%; outlier regions, 0% (Table 1). (Invitrogen, Carlsbad, CA, U.S.A.) with 10% fetal calf serum Effects of Chymotrypsin and Thermolysin Digestion (Bio West, Strasbourg, France) for 30 min at 37°C under 5% on the Activity of RNase Po1 and RNase T1 RNase Po1 CO2. The cells were collected by centrifugation at 1500 rpm and RNase T1 at 0.16 mg/mL were treated at a ratio of 1 : 17.5 and washed with PBS twice. The cell pellet was suspended (w/w) chymotrypsin : RNase in 10 mM Tris–HCl at pH 7.5 and in 0.1 mL of 0.5% Triton X-100 in 0.1 M Tris–HCl buffer (pH 37°C. RNase Po1 and RNase T1 at 0.14 mg/mL were treated 7.5) and frozen and thawed twice, then centrifuged again. The at 37°C with thermolysin at a ratio of 1 : 20 (w/w) thermo- RNase activity of the supernatant was measured as described lysin : RNase in 10 mM Tris–HCl at pH 7.5 containing 1 mM above and the RNase activity of the control was deducted (the

CaCl2. RNase activity in the reaction mixture was measured endogenous RNase activity of HL-60 cells). The relative rate as described above. of internalization was then calculated. Effect of RNase Po1 and RNase T1 with Cell-Pene- trating Peptide on the Proliferation of HL-60 Cells by RESULTS 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bro- mide (MTT) Assay Transfections of RNase Po1 and RNase Purification, Crystallization, and Determination of the T1 were performed according to the manufacturer’s instruc- Structure of RNase Po1 Expression of RNase Po1 in E. coli tions with cell-penetrating peptide (CPP) (TaKaRa Bio Inc., provided about 35000 units (approximately 17 mg) of RNase Shiga, Japan). The human leukemia cell line HL-60 was Po1 in 2 L of culture supernatant. RNase Po1 was homoge- purchased from the Health Science Research Resources Bank neous according to SDS-PAGE (Fig. 2). Purified RNase Po1

(Osaka, Japan). HL-60 cells were cultured in RPMI 1640 was crystallized with space group P31 and unit cell dimen- supplemented with 10% fetal calf serum. The cells were col- sions a=b=75.56 (Å), c=34.80 (Å). The structure of RNase lected by centrifugation and then suspended in in RPMI 1640 Po1 was solved using the Molecular Replacement method. The 6 without fetal calf serum and diluted to 1.5×10 cells/mL. One structure was refined at 1.85 Å resolution, with final Rwork and hundred and ten microliters of cell suspension was added to Rfree factors of 0.164 and 0.176, respectively. each well of a 96-well plate, and then 20 µL of 0.1 or 1.0 µM Atomic Structure of RNase Po1 The overall structure of final concentration of RNase Po1 or RNase T1 previously RNase Po1 is an (α+ β)-type structure consisting of an α-helix filtered through a Millipore filter (Millex GV, Billerica, MA, (residues 16–27) and seven β-strands (residues 7–9, 12–14, U.S.A.) was treated with Xfect Protein Transfection reagent 36–38, 52–56, 71–76, 82–86, 97–98), the same as RNase T1 for 30 min and added to the cells. Viable cells were counted (Figs. 3, 4). The refined model of RNase Po1 has an α-helix by MTT yellow tetrazole assay, after 18 h of incubation at with 3.5 turns, which is fewer than the helix in RNase T1

37°C under 5% CO2. After the addition of 10 µL of 0.5% MTT (4.5 turns). In RNase Po1, the helix runs like a “backbone” solution (Dojindo Laboratories, Kumamoto, Japan) to each down the molecule, and a four-stranded anti-parallel β-sheet well, incubation was continued for an additional 2–3 h at 37°C (36–38, 52–56, 71–76, 82–86) cross the α-helix. The catalyti- under 5% CO2. The absorbance at 630 nm was then measured. cally active amino acid residues of RNase T1 (His40, Glu58, Inhibition of cell proliferation was calculated as the percent Arg77) are located in the β3–6 strands (His40, Glu58, Arg77) decrease in final cell numbers as compared to the absence of and next to the β6 strands (His 92). It has also been reported RNase Po1 or RNase T1. for RNase T1 that in the transesterification step of phospho- Estimation of the Amounts of RNase Po1 or RNase T1 diester hydrolysis, His40 and/or Glu58 act as a general base Internalized into Tumor Cells The amounts of RNase Po1 toward the ribose 2′-hydroxyl group and that His92, as a gen- or RNase T1 in HL-60 cells were estimated using RNase ac- eral acid, donates a proton to the leaving 5′-hydroxyl group.5) tivity as a marker. Two milliliters of HL-60 cell suspension The catalytically active amino acid residues of RNase Po1 5 containing 3×10 cells/mL were incubated with 200 µL of 6 µM corresponding to RNase T1 are His36, Glu58, Arg72, and final concentration RNase Po1 or 6 µM RNase T1 or phosphate His87. These amino acid residues are also located in the β3–6

Fig. 4. Primary and Secondary Structures of RNase Po1 and RNase T1 Po1, RNase Po1; T1, RNase T1. Sequences in common are enclosed in boxes. Numbers above and below the matrix show RNase Po1 and RNase T1 numbering, respec- tively. The cysteine residues are shaded, and the disulfide bonds of RNase Po1 are shown as connected by bold lines. The catalytic site residues are indicated by arrows. Secondary structures are denoted as follows: α1, α-helix; βn, strand of β-sheet structure. 972 Vol. 37, No. 6 strands (His36, Glu58, Arg72) and next to the β6 strands (His RNase T1 (9 hydrophobic residues, 17 total). The hydrophobic 87). The base recognition site of RNase T1 consists of Tyr42, amino acid residue clusters of the β4–6 sheet of RNase Po1 Asn43, Asn44, Glu46, Tyr45, and Asn98 and is located in the are at opposite to the α-helix and may interact with those of loop between β3–4 strands (Asn43, Asn44, Tyr45, Glu46) and other strands and the α-helix, which then forms an internal in the loop between β6–7 strands (Asn98). The aromatic rings hydrophobic core similar to RNase T1 (Fig. 5). of Tyr42 (in the β3 strand) and Tyr45 stack with the guanine Disulfide Bonds in RNase Po1 RNase Po1 has six cys- base.5) In case of the base recognition site of RNase Po1, the teine residues. We determined the disulfide bond combina- amino acid residues Tyr38, Asn39, Asn40, Phe41, Glu42, and tions of these residues to be Cys9–Cys99, Cys7–Cys84, and Asn94 correspond to those of RNase T1. These amino acid Cys48–Cys82 (Fig. 6). One disulfide bond in RNase Po1 residues are also located in the loop between β3–4 strands (Cys9–Cys99) is superimposable on the analogous bond in (Asn39, Asn40, Phe41, Glu42) and in the loop between β6–7 RNase T1 (Cys6–Cys103). This disulfide bond is conserved strands (Asn94). The aromatic rings of Tyr38 (in the β3 in all known RNase T1 family enzymes, except for those strand) and Phe41 in RNase Po1 may stack with the guanine of bacterial origin. The locations of the other two disulfide base. Two important roles for the β-sheet in RNase T1 have bonds in RNase Pol (Cys7–Cys84 and Cys48–Cys82) do not been reported.2) One is that the β-sheet forms an internal correspond to any disulfide bond in RNase T1 (Fig. 4). The hydrophobic core, and the second is that the β-sheet structure Cys7–Cys84 bond is located parallel to the Cys9–Cys99 bond, forms the catalytic pocket. The three hydrophilic side chains thus making the C-terminus and N-terminus of RNase Po1 of Glu58, Arg77, and His92 in RNase T1 form a cluster on more rigid than those in RNase T1. The Cys48–Cys82 bond of the β-sheet surface opposite the α-helix and are involved in RNase Po1 is located near the active site on the opposite side enzymatic activity. The Glu54 (in the β4-strand), Arg72 (in of the Cys7–Cys84 bond. Moreover, the Cys48–Cys82 bond the β5-strand), and His 87 (next to the β6-strand) residues of connects the β6-strand to the loop between the β3-strand and RNase Po1 are conserved with them completely. The β4–6 β4-strand. In this loop, there are some amino acid residues sheet of RNase Po1 is inside the molecule and consists of 16 (Asn39, Asn40, Phe41, Glu42) thought to constitute the base amino acid residues, of which eight are hydrophobic, as in recognition site. Next to the β6-strand, there is one catalytic

Fig. 5. Hydrophobicity of the RNase Po1 Molecular Surfaces The figure was drawn with PyMOL (www.pymolwiki.org/index.php/Color_h). The molecules are shown as ribbons and surface models. The figure (b) is rotated horizon- tally by 180° by the center. βn, strand of β-sheet structure. The hydrophobic regions are colored gray and the deepness of the dark color shows hydrophobic strength. The circles enclose the hydrophobic core.

Fig. 6. The Electron Density Associated with the Disulfide Bonds Is Shown The cysteine residues and disulfide bonds are shown as sticks colored blue and yellow, respectively. The contour level is 1.2σ. βn, strand of β-sheet structure. (Color im- ages were converted into gray scale.) June 2014 973 residue (His87). Therefore, the Cys48–Cys82 bond may help than on the primary structure of the substrate. RNase Po1 maintain the conformational stability of the base recognition was more resistant to proteolysis than RNase T1, and RNase region and catalytic site (Fig. 3). Po1 retained 50–60% RNase activity after incubation with Effect of Chymotrypsin and Thermolysin Digestion on these proteases for 20 h, whereas RNase T1 retained only 15% the Activity of RNase Po1 and RNase T1 We digested RNase activity (Fig. 7). RNase Po1 and RNase T1 with chymotrypsin and thermoly- Effect of RNase Po1 and RNase T1 with Cell-Penetrat- sin to study their conformational stabilities. These proteases ing Peptide on the Proliferation of HL-60 Cells by MTT have broad specificity, so the proteolytic cleavage sites are Assay We transfected RNase Po1 and RNase T1 with CPP primarily determined by accessibility to the substrate rather and incubated them for 1 h. Viable cells were the same as for non-CPP cells. We then incubated them for 18 h, after which RNase Po1-CPP had decreased viable cells dependent on the RNase Po1 concentration, 20% (0.1 µM RNase Po1) or 60% (1.0 µM RNase Po1) lower than that of non-CPP cells. In contrast, RNase T1-CPP had a maximum of 10% decreased viable cells (1.0 µM RNase T1) (Fig. 8). We thus concluded that RNase Po1 caused greater inhibition of proliferation in HL-60 cells compared to RNase T1 after transfection. Estimation of the Amounts of RNase Po1 or RNase T1 Internalized into Tumor Cells We compared the amounts of RNase Po1 and RNase T1 in HL-60 cells internalized into HL-60 cells using RNase activity as a marker. The results are shown in Table 2. The RNase activity of RNase Po1 and RNase T1 deducted the endogenous RNase activity of HL-60 cells (7.0×10−3 units) by 17.78×10−3 units and 2.48×10−3 units, respectively. We calculated that the relative rate in HL-60 cells of RNase Po1 activity was four times higher than that of RNase T1. This suggested that RNase Po1 was internalized into HL-60 cells at a greater rate than RNase T1 and/or that RNase Po1 was considerably more stable in the cells.

DISCUSSION

There is high sequence identity (40%) between RNase Po1 and RNase T1, and complete conservation of the catalytic sequence.1) Both proteins are guanine-specific RNases with an optimum pH of 7.5 and with comparable specific activity towards RNA substrates. However, RNase Po1 exhibits anti- Fig. 7. Effect of Chymotrypsin and Thermolysin Digestion on the Ac- tumor activity towards several types of human tumor cells, tivity of RNase Po1 and RNase T1 whereas RNase T1 is non-toxic towards them.26) To investigate Digested RNase Po1 and RNase T1 by chymotrypsin (a) and thermolysin (b) and this difference, we determined the X-ray crystallographic measured RNase activity with reaction times. RNase activity just after starting the reaction was normalized to 100%. Circles show RNase Po1 and triangles show structure of RNase Po1 and compared it with that of RNase RNase T1. T1. RNase Po1 has an α-helix with 3.5 turns, which is shorter

Fig. 8. Effect of RNase Po1 and RNase T1 with Cell-Penetrating Peptide on the Proliferation of HL-60 Cells by MTT Assay HL-60 cells were treated with a given concentration of RNase Po1 or RNase T1 with or without cell-penetrating peptide (CPP). Viable cells were counted by MTT assay after 18 h of incubation at 37°C under 5% CO2. Cell proliferation without RNase was normalized to 100%. The graphs of −CPP show cell proliferation without CPP and are colored black, and those of +CPP show cell proliferation with CPP and are gray. The data represent the means and standard errors of three independent experiments, each performed in triplicate. (a), RNase Po1. (b), RNase T1. 974 Vol. 37, No. 6

Table 2. Internalization of RNase Po1 and RNase T1 in HL-60 Cells than the helix in RNase T1 (4.5 turns); thus, RNase Po1 has a more spherical shape (Fig. 3). The catalytic sites of RNase Po1 RNase activity RNase applied RNase activity in cells and RNase T1 are conserved, and each are composed of four RNase in cells to cells RNase applied to cells (units) (units) (%) β-strands (β3–6). A comparison of the molecular surfaces of RNase Po1 and RNase T1 clearly shows that although the −3 RNase T1 2.49×10 11.8 0.021 catalytic residues are conserved, the areas around the catalytic −3 RNase Po1 17.78×10 21.2 0.084 residues are poorly conserved (Fig. 9). A comparison of the

Fig. 9. Electrostatic Potentials of the RNase Po1 and RNase T1 Molecular Surfaces and Conserved Regions between Them The surface potential was calculated and displayed using the PyMOL ABPS tool.55) The central figures are viewed from the same direction as Fig. 3. The lower figures are rotated horizontally by 180° by the center. The circles enclose the active site. (a), (b) Electrostatic potentials of the RNase Po1 and RNase T1 molecular surfaces, re- spectively. The molecules are shown as ribbons and surface models. Negatively charged regions are shown in red, and positively charged regions are shown in blue. RNase T1 (a) is pink and RNase Po1 (b) is blue. (c) Conserved regions between the RNase Po1 and RNase T1 molecular surfaces. RNase Po1 is shown as ribbon and surface models. The conserved regions are colored orange and the others colored white. June 2014 975 hydropathy profiles54) of the amino acid sequences of RNase is guanine specific. The RNase activity of Onconase toward Po1 and RNase T1 shows that RNase Po1 is more hydropho- RNA is much lower than that of RNase Po1. The catalytic bic between residues 50–60 compared to RNase T1, because residues of Onconase consist of two histidines and one lysine two tyrosine residues (Y56, Y57) and one tryptophan resi- residue, and the sequence identity between Onconase and due (W59) of RNase T1 are changed to three phenylalanine RNase Po1 is very low.32) Onconase is a cationic protein that residues (F52, F53, F55) in RNsase Po1. In that region, there is contains a total of 15 positively charged residues (three argi- Glu 54 (RNase Po1) or Glu58 (RNase T1), part of the catalytic nines and twelve lysines), much more than RNase Po1 (eight site in the β4-strand (Figs. 4, 10). The molecular surface hy- arginines). The surface of Onconase has three high-density drophobicities of RNase Po1 and RNase T1 confirmed that the positively charged regions (patches) aside from the active site. hydrophobic region of RNase Po1 is larger than that of RNase These patches are very important in the liquid bilayer translo- T1 in the catalytic region, whereas the hydrophobicities of the cation step that is required for the cytotoxicity of Onconase.57) other regions are similar (Fig. 11). Hence, the catalytic region The three dimensional structure of Onconase39) is completely of RNase Po1 should be more stable than that of RNase T1 by different from that of RNase Po1; however, the electrostatic forming a wider hydrophobic region. We also compared the potentials of their molecular surfaces are similar in having electrostatic potentials of the molecular surfaces of RNase Po1 positively charged regions. This suggests that the positively and RNase T1.55) This showed that the surface of RNase Po1 charged regions of RNase Po1 might be important for binding is positively charged, but that the surface of RNase T1 is neg- to the plasma membrane of tumor cells electrostatically. ative, especially behind the catalytic site (Fig. 9). RNase Po1 α-Sarcin from Aspergillus giganteus has been reported to has eight arginine residues, much more than RNase T1 (one be a ribotoxin and contains the same active site of the RNase residue). Five of the eight arginine residues of RNase Po1 are T1 family.28) α-Sarcin is composed of a central antiparallel located at the molecular surface and make it positive. Since β-sheet packed against an α-helix (smaller than RNase Po1) cytotoxic RNases attack intracellular RNA, these RNases and has a conserved active site located on the other side, simi- must be internalized.41) Johnson et al.56) have suggested that a lar to RNase Po1.30) However, the sequence identity between family of cationic onconases, the RNase A family from Rana α-sarcin and RNase Po1 is low aside from the active site, and pipiens, which exhibit strong anti-tumor activity, bind to the α-sarcin has some insertion sequences with positive charges. plasma membrane electrostatically, and that enzyme binding One of them is a β-hairpin structure at the N-terminus (Leu7– is therefore critical to their anti-cancer properties. We com- Arg22), which suggests its involvement in interactions with pared RNase Po1 to Onconase in detail. The molecular weight the cell membrane, while the others are unstructured loops of Onconase is 12 kDa and the optimum pH toward RNA is that interact with the ribosomes58) (Fig. 12). RNase Po1 does pH 7.5, the same as RNase Po1. Therefore, the base specificity not have such structures. Thus, RNase Po1 likely binds to the of Onconase is pyrimidine base specific, whereas RNase Po1 plasma membranes of tumor cells by means different from that of α-sarcin. We earlier reported that RNase Po1 has higher thermal stability than RNase T1.1,26) The optimum temperature for catalysis was measured using RNA as a substrate at 20–80°C for 20 min. The optimum temperature for catalysis by RNase Po1 (70°C) was higher than that for RNase T1 (50°C). The

circular dichroism (CD) spectrum of RNase Po1 at [θ]210 nm,

Fig. 11. Hydrophobicity of the RNase Po1 and RNase T1 Molecular Surfaces Fig. 10. Hydropathy Profiles of the Amino Acid Sequences of RNase Po1 and RNase T1 The figure was drawn with PyMOL (www.pymolwiki.org/index.php/Color_h). RNase Po1 and RNase T1 are shown as surface models. The figures are viewed The hydropathy profiles were calculated by Expasy Tools (http://www.expasy. from the same direction as Fig. 5. The hydrophobic regions are colored gray and ch/tools/protscale html). Glu 54 (RNase Po1) and Glu58 (RNase T1) residues are the deepness of the dark color shows hydrophobic strength. 2′GMP is shown as denoted by arrows. sticks. 976 Vol. 37, No. 6

Fig. 12. Comparison of the Amino Acid Sequence of α-Sarcin with RNase T1 and RNase Po1 Po1: Pleurotus ostreatous RNase,1) T1: Aspergillus oryzae RNase,14) α-Sarcin: Aspergillus giganteus RNase.27) * Catalytic site. Disulfide bonds of RNases are shown con- nected by bold lines. The numbers at the top of the matrix are those of RNase Po1 and the bottom aero those of α-sarcin. The β-hairpin structure of α-sarcin is in italics. which reflects the peptide backbone conformation of the pro- superimposable with the Cys48–Cys82 bond of RNase Po1 tein, was maintained up to 60°C, then decreased very rapidly (Fig. 11). We found that RNase Po1 is likely internalized into with increasing temperature. In contrast, the [θ]210 nm value cells at a much greater rate than RNase T1 by comparing the of RNase T1 decreased in a biphasic manner, with breaks at RNase activities in tumor cells, and that RNase Po1 is prob- 40°C and about 60°C. The first sharp decrease around 40°C ably more stable in the cells than RNase T1 given the results corresponds to a decrease in the enzymatic activity of RNase of our proteolysis and cell transfection experiments. These T1. We transfected RNase Po1 and RNase T1 into HL-60 properties might be responsible for the inhibition of cell pro- cells. We found that RNase Po1 caused greater inhibition of liferation of RNase Po1. cell proliferation than RNase T1 (Fig. 8). The optimal pH of In conclusion, we investigated the X-ray crystallographic both enzymes was 7.5, and the specific activity of RNase Po1 structure of RNase Po1, which has anti-tumor activity, and for RNA was slightly higher than that of RNase T1; neverthe- found that it is more stable than RNase T1 because of an addi- less, RNase T1 might have inferior stability in cells. Prote- tional disulfide bond. Furthermore, RNase Po1 can bind to the olysis experiments showed that RNase Po1 is more resistant plasma membrane of tumor cells because its surface is posi- to chymotrypsin and thermolysin than RNase T1 (Fig. 7). tive, in contrast with RNase T1, which is negatively charged. This suggests that RNase Po1 may be more stable, even in These differences might contribute to the anti-tumor activ- human tumor cells. RNase Po1 contains six cysteine residues, ity of RNase Po1 towards human cancer cells. To construct two more than RNase T1. We investigated the disulfide bond RNase Po1 variants with stronger anti-tumor activity, we will combinations of these residues. One disulfide bond of RNase make RNase Po1 recombinants utilizing the information gath- Po1 (Cys9–Cys99) is conserved in all known RNase T1 family ered from this investigation. Further investigations into the enzymes from fungi. The other two disulfide bonds of RNase relationship between the structure and anti-tumor activity of Po1 (Cys7–Cys84 and Cys48–Cys82) do not have correspond- RNase Po1 may lead to the development of new anti-cancer ing bonds in RNase T1. The Cys7–Cys84 bond in RNase Pol drugs. connects the β1 to the β6 strand, and is located parallel to the Cys9–Cys99 bond, thus making the C-terminus and N- Acknowledgments We thank Professor Hiroshi Iijima for terminus of RNase Po1 more rigid. The Cys48–Cys82 bond helpful discussions. We also thank Dr. Ryuichi Kato and Ms. connects the β6 strand to the loop containing the recognition Taeko Sasaki for the crystallization of RNase Po1. This work site for the guanine base (Asn39, Asn40, Phe41, and Glu42).4) was supported by the Platform for Drug Discovery, Informat- Next to the β6-strand is the His87 residue which is part of the ics, and Structural Life Science from the Ministry of Educa- catalytic site. 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