Purification, crystallisation and preliminary X-ray diffraction study of ribosome inactivating protein: saporin

Biochimica et Biophysica Acta 1429 (1999) 506^511

Short crystallisation paper

Puri¢cation, crystallisation and preliminary X-ray di¡raction study of ribosome inactivating protein: saporin Mukesh Kumar *, Sharmishtha Dattagupta, K.K. Kannan, M.V. Hosur Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Received 16 September 1998; received in revised form 16 November 1998; accepted 24 November 1998

Abstract We report here the crystallisation and molecular replacement results on the structure determination of S-9 isoform of the ribosome inactivating protein saporin. The protein was purified to homogeneity by a simple and efficient protocol. The î , c = 150.66 A î and contain two molecules in the asymmetric crystals belong to the space group I41 with a = b = 91.47 A unit. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Ribosome inactivating protein; Crystal; X-ray; Structure

The ribosome inactivating proteins (RIPs) are plant toxins that arrest protein synthesis through depurination of a speci¢c adenosine in the highly conserved region of the ribosomal RNA [1^3]. It has recently been shown that most of the RIPs also deadenylate non-ribosomal substrates like poly(A), RNA, DNA and many di¡erent polynucleotides [4]. RIPs are classi¢ed into two groups. Type I RIPs contain a single polypeptide chain, while type II RIPs contain two polypeptide chains. Type II RIPs are actually synthesised as polyproteins, and posttranslational cleavage of an intervening sequence results in a protein with two polypeptide chains, A and B, linked by hydrophobic bonds and disul¢de bridges [2]. Chain B contains a lectin domain that interacts with the cell membrane and allows type II RIPs to enter the cell. Thus, type II RIPs are potent natural toxins, ricin being the best known example that has been used since ancient times for medicinal as well as

* Corresponding author.

criminal purposes [5^7]. Type I RIPs, on the other hand, lack the lectin domain and do not bind easily to cells. Consequently, they have a relatively low native cytotoxic activity. However for some cells, like macrophages [8] and trophoblasts [9,10], they are highly toxic, possibly due to high pinocytic activity of these cells. All type I RIPs have molecular weights in the range of 26^32 kDa, and have strong alkaline isoelectric points. There is growing interest in RIPs because of their potential therapeutic applications. They can be `glued' onto carrier molecules, like monoclonal antibodies forming immunotoxins [11], and also to hormones, growth factors or lectins which can be speci¢cally targeted against diseased cells [12,13]. Some of the important medical applications include treatment of cancer [14,15], HIV infection [16^18], parasitic [19] and autoimmune diseases [20]. It has been suggested that saporins would be ideal as the toxin component in immunotoxins due to the high stability of saporins against denaturation and proteolysis [21]. Several isoforms of a given RIP are isolated from

0167-4838 / 99 / $ ^ see front matter ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 2 6 7 - 2

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M. Kumar et al. / Biochimica et Biophysica Acta 1429 (1999) 506^511

Fig. 1. Puri¢cation of saporin isoforms through MonoS FPLC column using NaCl gradient (0^0.3 M). Loading bu¡er (A) is 50 mM NaOAc at pH 4.5 and elution bu¡er (B) is 2.0 M NaCl in 50 mM NaOAc at pH 4.5. (a) Elution pro¢le for saporin at pH 4.5. S-6 and S-9 fractions are labelled. (b) Rechromatography of the S-9 fraction showing its purity.

di¡erent tissues of plants, such as seeds, roots, leaves etc. Genomic analysis indicates that saporins are members of a multigene family [22,23] that encode for several isoforms that di¡er at speci¢c residues in the polypeptide chain. There is a great deal of variation in the deadenylation activity patterns of di¡erent isoforms of saporin towards di¡erent substrates and target cells. It was observed that the leave pro-

507

tein saporin-L2 is most active on Escherichia coli rRNA and poly(A), whereas the seed protein saporin-S9 is the most active on herring sperm DNA [4]. Saporin-L1 was the most active one on viral genomic RNAs of MS2, TMV and AMCV [4]. Detailed threedimensional structure is essential to understand the structure^function relationship of di¡erent isoforms as well as the reasons for the high stability of saporins. The three-dimensional structure is also highly desirable to engineer rational design of immunotoxins containing saporins. Crystallisation of isoform S-6 has been recently reported [24]. We report here our results on puri¢cation, crystallisation and molecular replacement solution of the isoform S-9. Seeds of Saponaria o¤cinalis (Soapwort) are a very rich source of saporins. Saporins account for nearly 10% of the total seed protein [25]. Saporin isoforms (mainly S-6 and S-9) were puri¢ed to homogeneity in two steps using MonoS cation exchange FPLC column (Amersham Pharmacia Biotech). The protocol is relatively simple and less time-consuming than previously reported methods [25^27], and gives high yields of very pure isoforms. Soapwort seed (25 g) was frozen in liquid nitrogen and crushed to ¢ne powder using a mortar and pestle. This powder was then extracted overnight in 200 ml of phosphate bu¡ered saline (pH 7.4) at 4³C. The extract was then centrifuged at 4000Ug for 1 h and the clear supernatant was dialysed extensively against 50 mM phosphate bu¡er at pH 6.5. Any precipitate found after dialysis was removed by centrifugation at 10 000Ug for 10 min. The clear solution was then loaded on a MonoS column equilibrated with 50 mM sodium phosphate bu¡er at pH 6.5, and the £ow-through fraction was found to contain the saporins. The other proteins bound to the column were washed out using 2.0 M NaCl in 50 mM phosphate bu¡er, pH 6.5. The £ow-through fraction was extensively dialysed against 50 mM sodium acetate bu¡er at pH 4.5. The clear ¢ltered solution was then loaded onto a MonoS column pre-equilibrated with 50 mM sodium acetate bu¡er at pH 4.5. Di¡erent isoforms of saporin were eluted out using 25 ml of 0^0.3 M NaCl gradient. Two major isoforms, S-6 and S-9 (Fig. 1a) eluted out at 0.20 M and 0.22 M NaCl. Typically a complete run took about 50 min. Rechromatography of S-9 fraction (Fig. 1b) shows very little contamination from S-6 fraction. Interest-

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Fig. 2. Crystals of saporin S-9.

ingly, the S-6 isoform could be obtained in pure form even without the ¢rst step of passage of crude extract through the MonoS at pH 6.5. This was not, however, possible for S-9. The ¢rst step of passing the crude extract through MonoS column at pH 6.5 was crucial for removing a contaminant that used to elute along with the S-9 isoform in the subsequent step at pH 4.5. This contaminant had the intriguing property of binding the MonoS column more strongly at pH 6.5 than at pH 4.5. The S-9 fraction was desalted and then concentrated to about 10 mg/ml with an Amicon's Centricon-10 concentrator. The extinction coe¤cient was assumed to be OM 280nm;1cm = 24 800 [26]. Crystallisation condition for S-9 was systematically explored by conventional hanging drop vapour di¡usion method [28] at room, temperature in 24-well cell culture plates. Protein solution (2 Wl) and reservoir solution (2 Wl) were mixed together and placed on a siliconised cover glass (18U18 mm). This was then inverted and sealed using vacuum grease on a reservoir containing 800 Wl of precipitant solution. Best crystals were obtained when the reservoir solution contained 0.15 M ammonium sulfate in 50 mM glycine at pH 9.5. Crystals were £at square plates of approximate size 0.2U0.2U0.06 mm3 (Fig. 2). X-ray di¡raction data on two such crystals were collected as 1³ oscillation frames on R-AXIS IIC

image plate detector mounted on Rigaku X-ray generator operating at 5 kW power and equipped with two focusing mirrors. The crystal to detector distance was kept at 100 mm. The crystals di¡racted up to î resolution (Fig. 3). Crystals were, however, 2.6 A found to be sensitive to X-ray radiation and the effective resolution after 2 h of exposure was limited to

Fig. 3. A typical di¡raction pattern of saporin S-9 crystal.

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Table 1 Molecular replacement using gelonin as search model AMoRea (1) Cross rotation function calculations

(2) One-body TF for rotation peak no. 1

Peak K

L

1 2 3

Peak K

L

143.8 21.4 15.9 36.8 203.0 8.9 64.6 103.7 7.8

1 2 3

118.46 129.71 0.1748U1031 7.25 83.39 136.12 0.1221U1031 5.07 91.81 155.98 0.1214U1031 5.04

Yfr:

Peak Xfr:

Yfr:

Zfr:

Dens

1 2 3

0.087 0.086 0.458

0.000 0.000 0.000

0.7975U1031 11.23 0.7734U1031 10.89 4.03 0.2859U1031

Peak Xfr:

Yfr:

Zfr:

Dens

1 2 3

0.087 0.018 0.960

0.750 0.949 0.317

0.6039 57.07 0.5944U1031 7.59 0.5935U1031 7.58

Xfr:

Yfr:

Zfr:

0.087 0.087

0.000 0.750

Zfr:

Cp

Rfactor CI

Yfr:

Zfr:

30.2 30.9 26.9

Rfactor CI

0.3238 0.7130 0.7500 47.9 0.9028 0.5464 0.5843 52.8 0.1159 0.5822 0.0512 53.1

54.9 32.4 31.9

After rigid body re¢nementc K

Molecule 1 Molecule 2

Q

0.8238 0.7131 0.0000 53.0 0.8258 0.2122 0.0000 53.0 0.4403 0.4901 0.0000 52.7

Peak Xfr: 1 2 3

(4) Final solution

9.3 27.8 1.8

Peak Xfr: 1 2 3

(3) Two body TF with molecule 1 ¢xed at 1-body TF solution

MolRepb

L

350.6 118.0 350.5 118.4

80.63 50.43 75.09

0.984 0.484 0.410

0.484 0.487 0.412

Q

RF

RF/ Sigma

Dens/ Sigma

Dens/ Sigma

Without re¢nementd

Q

Xfr:

Yfr:

Zfr:

K

L

Q

129.3 130.1

0.586 0.085

0.516 0.518

0.002 0.750

80.63 118.46 129.71 0.984 80.63 118.46 129.71 0.484

The ¢rst three peaks are listed for all types of calculation to show the contrast. All angles are in degrees. K, L and Q, Eulerian angles for rotation; CI , correlation of intensitiesU100; CP , truncated Patterson correlationU100; RF, rotation function value; Xfr: , Yfr: and Zfr: , fractional co-ordinates ; TF, translation function. a î ; integration radius, 35 A î. Initial rotation of the model, 191.75³, 29.50³, 287.35³; resolution range, 3.0^5.0 A b î ; Res_R, 3.0 A î ; integration radius, 35 A î. Initial rotation of the model, 0³, 0³, 0³; Resmax, 4.0 A c Correlation, 67.1%; Rfactor , 39.5%. d Correlation, 64.9%; Rfactor , 47.38%.

î . Di¡raction data were processed, scaled and 3 A merged together using the HKL software implemented on a SGI INDY graphics workstation [29]. The distortion parameter in the autoindexing procedure of DENZO showed very clearly the crystal lattice to be body centred tetragonal with unit cell diî and c = 150.66 A î . Laue mensions: a = b = 91.47 A symmetry was determined as 4/m on the basis of Rsym values. The calculated Vm value for 16 moleî 3 /Da which is within the cules in the unit cell is 2.76 A range of values observed in protein crystals [30]. Subsequently the molecular replacement calculation showed the space group to be I41 with two molecules in the asymmetric unit. For a total of 14 181 unique î resolution. re£ections Rsym value was 6% to 2.6 A î resolution The data was 74.4% complete to 2.6 A î and 97.7% complete up to 3.5 A resolution. Structure solution was attempted using the molec-

ular replacement method as implemented in the stand alone software packages AMoRe [31] and MolRep [32] on a Digital ALPHA workstation. Self-rotation function calculations showed only one two-fold peak along the z-axis which is a crystallographic two-fold symmetry axis in the space group I41 . Cross rotation function calculation was performed with the structure of gelonin [33], another type I RIP, as the search model. Both AMoRe and MolRep gave a single rotation peak with good contrast from the next highest peak as shown in Table 1. An n-body translation function search in MolRep gave an outstanding peak of height 57.07 times the standard deviation for two molecules. Corresponding correlation coe¤cient value in AMoRe was 54.9%. It is very interesting that the two molecules in the asymmetric unit have nearly identical orientation, and are related only by a simple translation of

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(1/2, 0, 3/4). Even though the ¢nal rotation and translation values are apparently di¡erent in AMoRe and MolRep (Table 1) they, however, generate the same set of molecules in the unit cell. Amino acid sequence of S-9 isoform is not yet available. Sequencing of the protein and crystalloî resolugraphic re¢nement of the structure at 2.6 A tion is currently in progress. The high resolution structure of S-9 would help understand the unusually high stability of saporins and also help to engineer highly e¤cient immunotoxins for therapeutic applications. We thank the DAE-DBT National Facility for Macromolecular Crystallography, SSPD, BARC, for the use of the biochemical equipment, data collection and computational facilities. We are also grateful to computer division for providing the computer facilities. We are grateful to Dr. Arun K. Mohanty and Ms. Bindu Pillai for many useful discussions. We are indebted to Prof. F. Stirpe, University of Bologna, Italy, for generous gift of saporin seeds.

References

[10]

[11] [12] [13]

[14] [15]

[16]

[17]

[18]

[1] W.K. Roberts, C.P. Selitrenniko¡, Plant proteins that inactivate foreign ribosomes, Biosci. Rep. 6 (1986) 19^29. [2] F. Stirpe, L. Barbieri, M.G. Batteli, M. Soria, D.A. Lappi, Ribosome-inactivating proteins from plants: present status and future prospects, Bio/Technology 10 (1992) 405^412. [3] Y. Endo, K. Mitsui, M. Motizuki, K. Tsurugi, The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and characteristics of the modi¢cation in 28 S ribosomal RNA caused by toxins, J. Biol. Chem. 262 (1987) 5908^5912. [4] L. Barbieri, P. Valbonesi, E. Bonora, P. Gorini, A. Bolognesi, F. Stirpe, Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: e¡ect on DNA, RNA and poly(A), Nucleic Acids Res. 25 (1997) 518^522. [5] S. Olsnes, A. Pihl, Molecular action of toxins and viruses, in: P. Cohen, S. Van Heyningen (Eds.), Elsevier, Amsterdam, 1982, pp. 51^105. [6] R.W. Apple, Pope case reminds Britons of unsolved Bulgarian crime, The New York Times 30 Dec (1982) A6-A0. [7] B. Knight, Ricin ^ a potent homicidal poison, Br. Med. J. 1 (1979) 350^351. [8] L. Barbieri, F. Stirpe, Ribosome-inactivating proteins from plants: properties and possible uses, Cancer Surv. 1 (1982) 490^520. [9] Y. Wang, R.-Q. Quian, Z.-W. Gu, S.-W. Jin, L.Q. Zhang, Z.X. Xia, G.Y. Tian, C.Z. Ni, Scienti¢c evaluation of Tian

[19]

[20]

[21]

[22] [23]

[24]

[25]

Hua Fen (THF) ^ history chemistry and application, Pure Appl. Chem. 58 (1986) 789^798. H.W. Yeung, W.W. Li, Z. Feng, L. Barbieri, F. Stirpe, Trichosanthin, K-momorcharin and L-momorcharin: identity of abortifacient and ribosome-inactivating proteins, Int. J. Pept. Protein Res. 31 (1988) 265^268. Y. Endo, in: A.E. Frankel (Ed.), Immunotoxins, Kluwer Academic, Boston, MA, 1988, pp. 75^89. D.A. Vallera, D.U. Myers, Immunotoxins containing ricin, Cancer Treat. Res. 37 (1988) 141^159. J.M. Lambert, W.A. Blattler, G.D. McIntyre, V.S. Goldmacher, C.F. Scott Jr., Immunotoxins containing single chain ribosome-inactivating proteins, Cancer Treat. Res. 37 (1988) 175^209. U. Brinkmann, I. Paston, Immunotoxins against cancer, Biochim. Biophys. Acta 1198 (1994) 27^45. E.J. Wowrzynczak, Systemic immunotoxin therapy of cancer: advances and prospects, Br. J. Cancer 64 (1991) 624^ 630. S. Lee-Huang, P.L. Huang, P.L. Nara, H.C. Chen, H.F. Kung, P. Huang, H.I. Huang, MAP30: a new inhibitor of HIV-I infection and replication, FEBS Lett. 272 (1990) 12^ 18. S. Lee-Huang, P.L. Huang, H.F. Kung, B.-Q. Li, P. Huang, H.I. Huang, H.-C. Chen, TAP-29: an anti-human immunode¢ciency virus protein from Trichosanthes kirilowii that is mitotoxic to intact cells, Proc. Natl. Acad. Sci. USA 88 (1991) 6570^6574. V.S. Byers, A.S. Levin, L.A. Waiter, B.A. Starrett, R.A. Mayer, J.A. Clegg, M.R. Price, R.A. Robins, M. Dalaney, R.W. Baldwin, A phase I/II study of trichosanthin treatment of HIV diseases, AIDS 4 (1990) 1189^1196. C.L. Villemez, P.L. Carlo, Preparation of immunotoxin for Acanthamoeba castellanii, Biochem. Biophys. Res. Commun. 125 (1984) 25^29. K.A. Krolick, Inhibition of autoimmune reactivity against acetylcholine receptor with idiotype-speci¢c immunotoxins, Methods Enzymol. 178 (1989) 448^458. S. Santanche¨, A. Bellelli, M. Brunori, The unusual stability of saporin, a candidate for the synthesis of immunotoxins, Biochem. Biophys. Res. Commun. 234 (1997) 129^132. A.P. Fordham-Skelton, A. Yarwood, R.R.D. Croy, Mol. Gen. Genet. 221 (1990) 134^138. I. Barthelemy, D. Martineau, M. Ong, R. Matsunami, N. Ling, L. Benatti, U. Cavallaro, M. Soria, D.A. Lappi, The expression of saporin, a ribosome inactivating protein from the plant Saponaria o¤cinalis, in Escherichia coli, J. Biol. Chem. 268 (1993) 6541^6548. C. Savino, L. Federici, A. Brancaccio, R. Ippoliti, E. Lendaro, D. Tsernoglou, Crystallization and preliminary X-ray study of saporin, a ribosome inactivating protein from Saponaria o¤cinalis, Acta Crystallogr. D54 (1998) 636^638. F. Stirpe, A. Gasperi-Campani, L. Barbieri, A. Falesca, A. Abbondanza, W.A. Stevens, Ribosome-inactivating proteins from the seeds of Saponaria o¤cinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and Asparagus o¤cina-

BBAPRO 30426 29-12-98

M. Kumar et al. / Biochimica et Biophysica Acta 1429 (1999) 506^511

[26]

[27] [28] [29]

lis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree), Biochem. J. 216 (1983) 617^625. J.M. Ferreras, L. Barbieri, T. Girbe¨s, M.G. Batteli, M.A. Rojo, F.J. Arias, M.A. Rocher, F. Soriano, E. Mende¨z, F. Stirpe, Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria o¤cinalis L. (Caryophyllaceae), Biochim. Biophys. Acta 1216 (1993) 31^42. G. Marcozzi, L. Spano, A simple method for the puri¢cation of type I RIPs, Biomed. Chromatogr. 11 (1997) 151^153. A. McPherson, in: Puri¢cation and Analysis of Protein Crystals, John Wiley and Sons, 1982, New York. Z. Otwinowski, Oscillation data reduction program, in: Pro-

[30] [31] [32]

[33]

511

ceedings of the CCP4 study weekend: `Data Collection and Processing', compiled by L. Swyer, N. Isaacs, Bailys SERC Daresbury Laboratory UK, 1993, pp. 56^62. B.W. Methews, Solvent content of protein crystal, J. Mol. Biol. 33 (1968) 491^497. J. Navaza, AMoRe: an automated package for molecular replacement, Acta Crystallogr. A50 (1994) 157^163. A. Vagin, A. Teplykov, MOLREP : an automated program for molecular replacement, J. Appl. Crystallogr. 30 (1997) 1022^1025. M.V. Hosur, B. Nair, P. Satyamurthy, S. Misquith, A. Surî resolia, K.K. Kannan, X-ray structure of gelonin at 1.8A olution, J. Mol. Biol. 250 (1995) 368^380.

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