THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 1, Issue of January 5 , pp. 245-251,1991 Printed in U.S.A.

Amino Acid Sequence of an Anti-tumor Protein from Rana pipiens Oocytes and Early Embryos HOMOLOGY TO PANCREATIC RIBONUCLEASES*

(Received for publication, July 25, 1990)

Wojciech ArdeltS, Stanislaw M. Mikulski, and Kuslima Shogen From the Alfacell Corporation, Bloomfield, New Jersey 07003

Rana pipiens oocytes and early embryos contain large amounts of a basic protein with antiproliferativel cytotoxic activity against several tumor cell lines in vitro (Darzynkiewicz, Z., Carter, S. P., Mikulski, S. M., Ardelt, W., and Shogen, K. (1988) Cell Tissue Kinet. 21,169-182; Mikulski, S. M., Viera, A., Ardelt, W., Menduke, H., and Shogen, K. (1990) Cell Tissue Kinet. 23, 237-246), as well as antitumor activity in vivo (Mikulski, S. M., Ardelt, W., Shogen, K., Bern- stein, E. H., and Menduke, H. (1990) J. Natl. Cancer Inst. 82, 151-153). The protein, provisionally named P-30 Protein, was purified to homogeneity from early embryos and characterized. It is a single-chain protein consisting of 104 amino acid residues in the following sequence: <Glu’-Asp-Trp-Leu-Thr-Phe-Gln-Lys-Lys- His-Ile-Thr-Asn-Thr-Arg”-Asp-Val-Asp-Cys-Asp- Asn-Ile-Met-Ser-Thr-Asn-Leu-Phe-His-Cys30-Lys- Asp-Lys-Asn-Thr-Phe-Ile-Tyr-Ser-Arg-Pro-Glu-Pro- Val-Lys46-Ala-Ile-Cys-Lys-Gly-Ile-Ile-Ala-Ser-Lys- Asn-Val-Leu-Thr-Three-Ser-Glu-Phe-Tyr-Leu-Ser- Asp-Cys-Asn-Val-Thr-Ser-Arg-Pro-Cys7S-Lys-Tyr- Lys-Leu-Lys-Lys-Ser-Thr-Asn-Lys-Phe-Cys-Val- Thr-Cys90-Glu-Asn-G1n-Ala-Pro-Val-His-Phe-Val- Gly-Val-Gly-Ser-Cy~’~~-OH. Its molecular weight cal- culated from the sequence is 11,819. The sequence homology clearly indicates that the protein belongs to the superfamily of pancreatic ribonuclease. It is also demonstrated that it indeed exhibits a ribonucleolytic activity against highly polymerized RNA and that this activity seems to be essential for its antiproliferativel cytotoxic effects.

Based upon the original in vivo work of Shogen and Yoan,‘ it was found that extracts of Rana pipiens early embryos (up to four blastomere stage of development) exert an antiproli- ferative/cytotoxic activity against numerous cancer cell lines in vitro (Darzynkiewicz et al., 1988). Therefore, an effort was undertaken to isolate and characterize the active compo- nent(s). It was then found that a basic protein present in large quantities in the source material was responsible for most of the activity. At least two other active proteins of much lower content in the extracts were also detected. The major protein (provisionally named P-30 Protein) was isolated and its activ- ity against cancer cell lines studied in vitro (Darzynkiewicz et

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ Presented at the 27th Annual Eastern Colleges Science Confer- ence, Pennsylvania State University, April 28, 1973 (K. Shogen and W. K. Yoan, unpublished study).

al., 1988; Mikulski et al., 1990b). It has also been demonstrated that the protein has a substantial antitumor effect in vivo (Mikulski et al., 1990a).

In this paper we present the complete amino acid sequence of this protein, as well as the purification procedure leading to its homogeneous preparation from either early embryos or oocytes. We also demonstrate that the protein is highly ho- mologous to pancreatic ribonucleases and exhibits a ribonu- clease-like activity against high molecular weight ribosomal RNA. This activity may be critical for the anti-tumor effects of P-30 Protein. Further enzymatic characterization of this protein is in progress in our laboratory.2

EXPERIMENTAL PROCEDURES3

RESULTS

Sequencing Strategy-Sequencer runs of intact as well as of reduced and pyridylethylated P-30 Protein (RPE4-P-30 Protein) (100 nmol, spinning cup sequenator) gave no PTHs, indicating a blocked NH2-terminus. The RPE-derivative was therefore subjected to fragmentation with CNBr, or Staphy- lococcus aurew proteinase V-8, and the citraconylated RPE- derivative was digested with trypsin to generate overlapping peptides. NH2-terminal (blocked) peptides were treated with pyroglutamate aminopeptidase. This resulted in removal of the NH2-terminal pyroglutamic acid residue and allowed us to sequence the fragments starting from the second residue. Detailed documentation of the generation and analysis of the peptides is given in the miniprint section.

Alignment of Peptides-This is presented in Fig. 1. Two CNBr fragments, four tryptic peptides and four fragments generated by S. aureus proteinase V-8 were aligned to rigor- ously prove the complete sequence of the 104 residues of P- 30 Protein. Amino acid compositions of the entire protein and of all peptides used (Tables Sll. and SIII.) are in very good agreement with the deduced sequence. The NH2-terminal pyroglutamic acid was the only residue determined indirectly. The proof is based on the residue removal by pyroglutamate aminopeptidase (thus unblocking the NH2 terminus) and on

The protein manufactured by Alfacell Corporation, Bloomfield, NJ, is currently undergoing clinical trials in the United States.

Portions of this paper, (including “Exprimental Procedures,” part of “Results,” Tables SI-SVI, and Figs. S1-S5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: RPE, reduced and pyridylethylated; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- resis; HPLC, high performance liquid chromatography; RP, reverse- phase; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; DTT, dithiothreitol; PE-Cys, pyridylethylcysteine.

245

246 Sequence of R. pipiens Oocyte Anti-tumor Protein

10 20 30 40 50 60 70 80 90 100

CEDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

I- CNBr-1 CNBr-2

-I: """"""""""""""""" -I

T-1 1 " 1

T-2 """""""""_""""""""""""""""""""""""~ -I

T-3 T-4 I """""" -I1 I

SP-1 SP-2 I""-"""""""""""""""""""""""""""" -I,"""""""""""- 4

SP-3 I -I-

SP-4

FIG. 1. Alignment of the peptide fragments of R. pipiens oocyte anti-tumor protein. The om-letter amino acid notation is used ( 4 3 represents pyroglutamic acid). CNBr-1 and CNBr-2, fragments generated by cyanogen bromide cleavage; T-1 to 7'-4, tryptic peptides generated from citraconylated ribonuclease; SP-1 to SP- 4, peptides generated by staphylcoccal proteinase V-8. Solid horizontal lines represent actually sequenced segments; short vertical lines indicate peptide ends; broken lines represent peptide regions that were not sequenced but are consistent with the generated sequence as judged from amino acid analysis.

amino acid analysis of the NH2-terminal fragments before and after the digestion. Among the remaining 103 residues, S e P , G1u6', and Glugl were determined in single runs only, other residues were identified at least twice in different pep- tides. A carboxyl-terminal cysteine residue (as PE-Cys) was found in two peptides generated by two proteinases of quite different specificity. Neither staphylococcal proteinase nor trypsin were expected to hydrolyze peptide bonds formed by PE-Cys and, indeed, none of the seven PE-Cys-X bonds located earlier in the chain was cleaved. COOH-terminal Cys was also independently determined by hydrazinolysis of the oxidized derivative of the protein (cysteic acid was found). These results make the evidence for the COOH-terminal residue stringent.

The molecular weight of P-30 Protein calculated from the sequence, assuming that all Cys residues are paired, is 11,819.

Ribonucleolytic Activity-As we discuss later, the sequence was found to be similar to that of RNase A. Therefore, we decided to look for a potential RNase-like activity in the protein. We found it active against highly polymerized ribo- somal RNA. The specific activity values were 1.85 X lo3 and 1.0 X lo3 activity units/mg of P-30 Protein, as tested against yeast and wheat germ RNA, respectively. Corresponding val- ues for RNase A were 1.0 X lo6 and 7.1 X lo6 units/mg, respectively. Therefore, it is evident that the protein seems to be several hundred times less active toward these substrates than RNase A. To test the possibility that the observed ribonuclease-like activity is due to contamination with a highly active RNase, we ran P-30 Protein on a reverse-phase HPLC column (Fig. 2). The main peak representing more than 90% of the material loaded on the column, as well as two more retarded small peaks, retained full biological (anti- tumor), and RNase-like activities. Each of the three peaks demonstrated the same amino acid composition, matching exactly that of P-30 Protein (presented in Table SII). It seems, therefore, that the three peaks represent different conforma- tional forms of the same protein. The two more retarded peaks could be the products of reversible denaturation of P- 30 Protein. Similar results were reported earlier for RNase A (Cohen et al., 1985). Our experiment demonstrated that the preparation of P-30 Protein that was homogeneous according to criteria of size, charge, and hydrophobicity is active against tumor cells, as well as against highly polymerized ribosomal

0.15

0 ' 0 RETENTION TIME (MIN)

FIG. 2. Reverse-phase HPLC of P-30 Protein. Two mg of the protein were loaded onto an Altex Ultrasphere-ODs, 5 pm, 10 X 250- mm column. The column was developed with a 14-35% (v/v) gradient of 1-propanol in 0.1% (v/v) trifluoroacetic acid. The fractions indi- cated by solid bars were isolated and lyophilized.

20 40 66

RNA. Therefore, it is highly probable that the two activities are inherent properties of the same molecule.

We also repeated the classic experiment of Crestfield et al. (1963) on RNase A, to inactivate the RNase-like activity of the P-30 Protein. Alkylation of the protein with iodoacetic acid (which resulted in modification of the catalytic histidine residues in RNase A), almost completely abolished the RNase- like activity of P-30 Protein (4% remaining activity against yeast RNA). The alkylated preparation was also inactive against A-253 human squamous carcinoma cells in vitro. This indicates that the ribonucleolytic activity of our protein may be essential for its antiproliferative/cytotoxic effects.

Lack of Agglutinating Actiuity-Since the closest sequence similarity was found between P-30 Protein and a sialic acid- binding lectin from bullfrog oocytes (see "Discussion" and Fig. 3), we tested the protein for potential cell agglutinating activity, which is characteristic for lectins. Human erythro- cytes and three tumor cell lines, A-253 human squamous carcinoma, human pancreatic ASPC-1 adenocarcinoma, and mouse Ehrlich ascites carcinoma were tested. No agglutinin- like effect was detected.

DISCUSSION

The present study was undertaken to characterize an anti- proliferative/cytotoxic activity against some tumor cell lines

Sequence of R. pipiens Oocyte Anti-tumor Protein 241

10 ~ ~ 20 30

FIG. 3. Alignment of the sequence of R. pipiem oocyte anti-tumor pro- tein (P-30 Protein) with those of bo- vine pancreatic ribonuclease A (Smyth et al., 1963), bovine angi- ogenin (Bond and Strydom, 1989), and Rana catesbiana (bullfrog) sialic acid binding lectin (Titani et al., 1987). Residues are numbered ac- cording to bovine RNase A. Identities between P-30 Protein and either bullfrog lectin, bovine angiogenin, or bovine RNase A are boxed. Catalytic residues of bovine RNase A are marked by solid circles and other residues forming the active site of this enzyme, by open circles.

Bovine RNase A

B u l l f r o g l e c t i n P-30 P r o t e i n

B o v i n e a n g i o g e n i n

4 0 . 0 0 0 50 60

B o v i n e a n g i o g e n i n B u l l f c o g l e c t i n

Bovine RNase A

P-30 P r o t e i n

70 - so 90 B o v i n e R N a s e A

B u l l f r o g l e c t i n B o v i n e a n g i o g e n i n

P-30 P r o t e i n

O D

1 1 0 Bovine RNase A N P Y V I P V H FlD A S \

. l P o 0

Bovine angiogenin B u l l f r o g l e c t i n P-30 P r o t e i n

first encountered in early embryos and then in unfertilized oocytes of R. pipiem. The major active protein purified from early embryos was found homologous to pancreatic RNases, angiogenins and a sialic acid-binding lectin (Fig. 3). This sequence homology was totally unexpected. The next finding was less surprising. We demonstrated that our protein pos- sesses an RNase-like activity toward highly polymerized RNA. We also present some indirect evidence that antipro- liferative/cytotoxic and RNase-like activities may be inherent properties of the same molecule, and that the latter activity may be essential for the biological effects of P-30 Protein.

The presented amino acid sequence was rigorously proven (Fig. 1). A set of overlapping peptides generated by chemical and enzymatic cleavages was used, and their sequences were confirmed by amino acid analysis (Tables SI1 and SIII). NHz- terminal pyroglutamic acid was determined by its removal with a specific enzyme and amino acid analysis. One-hundred residues (96%) were determined more than once by sequenc- ing different overlapping fragments. The COOH-terminal res- idue was confirmed by hydrazinolysis.

The sequence was compared to all sequences registered with the Protein Identification Resource, National Biomedical Re- search Foundation, and was found to be unique. Its relation- ship to pancreatic RNases was simultaneously demonstrated. The protein seems to be the smallest member (104 residues) of this superfamily. Fig. 3 shows that it is 53% identical with sialic acid-binding lectin, 31% identical with bovine angi- ogenin, and 30% identical with RNase A. Respective numbers for three other proteins not listed in Fig. 3 are 29% for human angiogenin (Strydom et al., 1985), 27% for bovine seminal plasma RNase (Di Donato and D'Alessio, 1973), and 22% for snapping turtle RNase (Beintema et al., 1985).

All 3 catalytic residues of RNase A, His", Lys41, and His'lg are strictly conserved in P-30 Protein (Fig. 3) at the homolo- gous positions (His", Lys3', and Hisg7), as well as in other sequences aligned in the figure. Another 4 active site residues of RNase A, at positions 4445, 111, and 120, have identical counterparts in the protein (positions 34, 35, 91, and 98, respectively). Gln6' is either replaced by Leu in P-30 Protein or deleted (the gaps in this region could be positioned differ- ently). It is also deleted in angiogenins, bullfrog lectin, and turtle RNase. A d 1 is deleted in P-30 Protein, bovine angi- ogenin (but not in human angiogenin), the lectin, and the turtle enzyme. In RNase A, a putative role of these two residues is the formation of hydrogen bonds from their side chains to purine nitrogen atoms (Wodak et al., 1977; Shapiro et al., 1986). It is therefore possible that their absence in P-

30 Protein and angiogenins results in the lower RNase-like activities and/or different specificities of these proteins, as compared with RNase A. Another 3 residues of the active site of RNase A, Gln", Asp1'', and Serlz3 are conserved in pan- creatic RNases and angiogenins, but not in the protein de- scribed here or the lectin. Gln" is replaced by Lys, Asn'", and Serlz3 by neutral residues in the last two proteins. Thus, at least two hydrogen bonds formed during RNase A-catalyzed reactions, i.e., from the y-carboxyl group of Asp'" to His"' and from the hydroxyl group of SerlZ3 to the uracil oxygen (Shapiro et al., 1986), could not be formed in the interaction of this protein or that of the lectin, with RNA. These changes may also contribute to the apparent differences in biological activity between P-30 Protein and other members of the superfamily. Residues forming a "non-polar cluster'' (Carlisle et al., 1974; Lewis et al., 1989) in RNase A are conserved among pancreatic RNases, angiogenins and, with the excep- tion of Ilelo7, in bullfrog lectin (Lewis et al., 1989), and in P- 30 Protein. In the last two proteins this residue is replaced by cysteine.

Disulfide bridges were not determined in the present study. However, P-30 Protein (like angiogenins, turtle RNase, and bullfrog lectin) contains 6 Cys residues in positions identical to those in RNase A (Fig. 3). It is, therefore, predictable that three of the four disulfide bridges of RNase A at positions 26- 84,40-96, and 58-110 are conserved as 19-68,30-75, and 48- 90 in this protein. The fourth disulfide bond of RNase A, between and Cys7', present in all known pancreatic RNases (except for the snapping turtle enzyme) is missing in P-30 Protein, angiogenins, and bullfrog lectin. In RNase A, this disulfide bridge forms an exposed loop containing the previously discussed Gln6' and Asn7' (a component of the purine-binding site). The importance of a region of this di- sulfide linkage for the biological activity of angiogenins was recently demonstrated by Harper and Vallee (1989).

P-30 Protein and bullfrog lectin contain 2 cysteine residues absent from known RNases. These are Cysa7 and Cys'04 in this protein (positions 107 and 126 in RNase A, Fig. 3). Amino acid analysis of P-30 Protein hydrolyzed without dithiodipro- pionic acid (not shown here), indicated that all Cys residues are paired. Thus, it is highly probable that Cysa7 and Cys'04 form the fourth disulfide bridge, characteristic for this protein and the lectin. In that case, a COOH-terminal loop is gener- ated. The COOH-terminal Cys (in our protein) would be placed in close vicinity of the "hydrophobic cluster" inside the active site cleft. This, in turn, could affect the enzymatic properties of the protein and its biological activity.

248 Sequence of R. pipiens Oocyte Anti-tumor Protein

Lewis et al. (1989) suggested that bullfrog lectin should have a ribonucleolytic activity as the protein is highly similar to RNases. Our studies support this hypothesis as P-30 Pro- tein (53% identical with the lectin) is active against RNA. Bullfrog lectin inhibits tumor growth in vitro (Nakaima et al., 1986) and i n vivo (unpublished results discussed by Titani et al., 1987). These authors speculated that these effects are mediated through immune modulation. P-30 Protein does not share agglutinating properties with the lectin. On the other hand, its antiproliferative/cytotoxic activity against A-253 squamous carcinoma cells, as well as its RNase-like activity, are abolished by modification with iodoacetic acid. Therefore, it seems probable that RNase-like activity is critical for the anti-tumor effect of the protein.

The physiological significance of the P-30 Protein is un- known, as is the mechanism of its biological activity. In several tumor cell lines the protein arrests cells in the GI- phase of the cell cycle (Darzynkiewicz et al., 1988). Within the range of concentrations used against tumor cells, the growth of all of the studied normal human cells, such as skin fibroblasts, keratinocytes, and peripheral blood lymphocytes' was not significantly affected.

Acknowledgments-Gas sequenator runs were performed by H. Lackland at the Center for Advanced Biology and Medicine, Pisca- taway, NJ (Laboratory of Dr. S. Stein). Spinning cup sequenator runs and most of the amino acid analyses were carried out in the Depart- ment of Chemistry, Purdue University, West Lafayette, IN (Labora- tory of Dr. M. Laskowski, Jr.). We wish to thank Dr. I. Apostol of Purdue University for many helpful discussions. We wish to acknowl- edge the skillful technical assistance of M. Morningstar, M. Piesla, J . Riley, C. Spence, and A. Viera, all of Alfacell Corporation.

REFERENCES

Akabori, S., Ohno, K., and Narita, K. (1952) Bull. Chem. SOC. Jpn

Anfinsen, C. B., Redfield, R. R., Choate, W. L., Page, J., and Carroll,

Barkholt, V., and Jensen, A. L. (1989) Anal. Biochem. 177,318-322 Beintema, J. J., Broos, J., Meulenberg, J., and Schuller, C. (1985)

Blank, A., and Dekker, C. A. (1981) Biochemistry 20,2261-2267 Bond, M. D., and Strydom, D. J. (1989) Biochemistry 28,6110-6113 Bradbury, J. H. (1956) Nature 178,912-913 Carlisle, C. H., Palmer, R. A., Mazumdar, S. K., Gorinsky, B. A., and

Cohen, S. A., Benedek, K., Tapuhi, Y., Ford, J. C., and Karger, B. L.

25, 214-218

W. R. (1954) J. Biol. Chen. 207, 201-210

Eur. J. Biochem. 153,305-312

Yeates, D. G. R. (1974) J. Mol. Biol. 85. 1-18

(1985) Anal. Biochem. 144, 275-284 Crestfield. A. M.. Stein. W. H.. and Moore. S. (1963) J . Biol. Chem.

238, 2413-2420

Shogen, K. (1988) Cell Tissue Kinet. 21, 169-182

, . .

Darzynkiewicz, Z., Carter, S. P., Mikulski, S. M., Ardelt, W., and

Di Donato, A., and D'Alessio, G. (1973) Biochem. Biophys. Res.

Dixon, H. B. F., and Perham, R. N. (1968) Biochem. J . 109,312-314 Fontana, A., and Gross, E. (1986) in Practical Protein Chemistry: A

Handdbook (Darbre, A., ed) pp. 67-120, John Wiley & Sons, New York

Harper, J. W., and Vallee, B. L. (1989) Biochemistry 28, 1875-1884 Hermodson, M. A., Ericsson, L. H., Neurath, H., and Walsh, K. A.

Hewick, R. M., Hunkapillar, M. W., Hood, L. E., and Dreyer, W. J.

Hoy, T. G., Ferdinand, W., and Harrison, P. M. (1974) Znt. J . Pept.

Hunkapillar, M. W., and Hood, L. E. (1983) Methods Enzymol. 91,

Kawauchi, H., Sakakibara, F., and Watanabe, K. (1975) Experientia

Laemmli, U. K. (1970) Nature 227, 680-685 Lewis, M. T., Hunt, L. T., and Barker, W. C. (1989) Protein Seq.

Lottspeich, F. (1980) Hoppe-Seyler's 2. Physiol. Chem. 361, 1829-

Mahoney, W. C., Smith, P. K., and Hermodson, M. A. (1981) Bio-

Mikulski, S. M., Ardelt, W., Shogen, K., Bernstein, E. H., and

Mikulski, S. M., Viera, A., Ardelt, W., Menduke, H., and Shogen, K.

Mosmann, T. (1983) J. Immunol. Methods 65,55-63 Nakijama, Y., Suzuki, H., Sakakibara, F., Kawauchi, H., Mizuno, D.,

and Yamazaki, M. (1986) Jpn. J. Exp. Med. 56, 19 Podell. D. N.. and Abraham. G. N. (1978) Biochem. Biophvs. Res.

Commun. 55,919-927

(1973) Biochemistry 12, 3146-3153

(1981) J . Biol. Chem. 256, 7990-7997

Protein Res. 6, 121-140

486-493

3 1,364-365

Data Anal. 2, 101-105

1834

chemistry 20,443-448

Menduke, H. (1990a) J. Nat. Cancer Inst. 82, 151-153

(1990b) Cell Tissue Kinet. 23, 237-246

Commun. 81,176-185 '

. . _ _ ShaDiro. R.. Riordan. J. F.. and Vallee. B. L. (1986) Biochemistry 25,

, I

3527-3532 Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner,

F. H., Provenzano, M. D., Fujimoto, E. K., Gaeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,76-85

Smyth, D. G., Stein, W. H., and Moore, S. (1963) J. Biol. Chem. 238,

Strydom, D. J., Fett, J. W., Lobb, R. R., Alderman, E. M., Bethune, J. L., Riordan, J. F., and Vallee, B. L. (1985) Biochemistry 24,

Titani, K., Takio, K., Kuwada, M., Nitta, K., Sakakibara, F., Kawau- chi, H., Takayanagi, G., and Hakomori, S. (1987) Biochemistry 26, 2189-2194

Wodak, S. Y., Liu, M. Y., and Wyckoff, H. W. (1977) J. Mol. Biol.

. .

227-234

5486-5494

116,855-875

Sequence of R. pipiens Oocyte Anti-tumor Protein 249

Olgest~on with S . aureus Proteinase V - 8 . RPE-P-30 Proteln 1 4 0 0 nmolesl *as Incubated with the enzyme 1200 p g l In 1 . 1 m i of 0.05M ammonium acetate. pH 4 . 0 for 8 h at 23'C. The mixture !;as acldlfled to DH 2 . 0 with HCI and

Hlsh Performance Liquid Chromat.oy(raohx. Homogeneity of the peptide fragment3 was tented on anslytlcal, reverse phase HPLC. Sone peptides aeparated by Size exclusion chromatography were further purlfied by HPLC before

columns were used. The column% were d e v ~ l o P ~ ' b 7 various aradlents p~epared sequencing. The A l t e x Ultraaphere-ODS 5 4 . 6 x 150 m m lor 10 x 250 m m l

from 0.1% TFA laalvent A I and 0.1% TFA. 5 0 % 1-propanol (solvent 81.

Amlno Acid A n a l y s e s . Proteln samples were hydrolyzed in 6H HCl containing

w a s carrled out m, ~n sealed 9 m tubes at llO°C for 1 2 . 24. 4 8 and 7 1 0.05% 3,3'-dithiod,propionlc acid IBerkholt end Jensen. 1 9 8 9 1 . Hj-drolysis

buffer and run ( 0 . 7 5 - 4 , 1 nmolesl On a Beckman 7 3 0 0 Amlno Acid Analysis h. The rube contents were drlcd under YaCYum, reconstltuted ~n the pH 2 . 2

System. A computer integer fit program. after Hoy et ai. 1 1 9 7 4 l . crrlculsted the results. Peptlde fragments -ere hydrolyzed for 21 h uzthout dlthiodiproplonlc ecld.

Sequence Anelvses. M o s t of the automated amlno ecld sequence analyses were performed on an Applied Blasyetems, 1 ° C . . 4 7 O A Prateln Sequencer (Hewick et al., 19811 vlth 120A on-ilne PTH Amino Acld Analyzer IHunkspillar and Hood, 19831 and a 9 O O A ControlIDeta 4nalyrls Module. The runs ere recorded and results calculated using an AB1 1 1 5 A Report Generator. Some early sequencer rung were carrled out on 1 Beckman 89OC spinning-cup sequencer equipped vlth a Sequenat P6 converter. A 90-mln Quadrol program Vas employed. Hundred nlnolen proteln samples were run and PTH-amino aclds were identified by

determined by hydrszlnolysis Irlkabarl et el., 1 9 5 2 : Bradbury, 1 9 5 6 1 . lsocratic HPLC ILottspezch, 19801. The carboxyl terminal residue w a s

Oxidized P-30 Protein (25 nrooles) was treated with anhydrous hydrazine for 18 h st 8O'C. Hydrazine w a s removed by lyaphilirstlon. The sample w a s reconstltuted in a pH 2 . 2 buffer and run on an amino acld analyzer.

Anri~ro l i f erar iveICyto tDxic A c t i v i t ~ , 1-253 human squamous ~erclnoma c e l l line was used. The cello 1 2 4 , 0 0 0 per well, in 100 ulJ were incubated wlth Various concentrations (10 ng tn 10 pg/m11 of P-30 Proteln l o r RHaae A I at 37'C for 7 2 h. in a cell culture medium consisting of RPMI 1640 with 10%

cella YIP determined I" each well by a colorlnetrlc assay for cell survival fetal bovine 8 e w m Inazletan, Lenexa, Kan~asl. The fractxon of surviving

Chenicon, Tenecula. Callfornla. and proliferation. as described by Hoalnann 1 1 9 8 3 ) . uslnz a reagent kit of

Inactivation of P-30 Protein. The enzyme was alkylated bv e madiflcatlon of the method of Crestfield. Steln and Moore 119631. The prateln 12.5 mg in 0 . 2 m l of 0.05'1 sodium acetate buffer. pH 5 . 5 was incubated with 6.2 ma of iodoacetic acid sodium salt for 8 h at 23.C. then desalted on a Blo-Gel P-2

Cell Auulutinatian A s s a y . Thls was performed eccordrng to the method of Bavauchi. Sakaklbara and Watanabe 1 1 9 7 5 ) . All cell ilnes used were from the American Type Culture Collection. Rockville, Maryland.

RESULTS

Purification of Rana ~ l p l e n s oocyte antl-tumor oroteln. F l g s . S . l . , S . 2 A . and 5.28. illustrate e three step p~rlf~catlon procedure. lost of the inactive material w a s eluted k n s large breakthrough fracrlon durlng the ion exchange chromatography (Fig. S . 1 . I . M l J O P portion of the antlprollferat~ve /cytotoxic actlvity w a g found in the third 1 m e ~ o r I peek eluted u ~ r h the salt %=adient. S u b s t a n t l a 1 amount of the sctlvlty we= a150 detected ~n the two minor peaks eluted later in the arsdlent lmore baslcl. Only the lna~or active peek was used for further puriflcst~on I" this study; the actlve c o m o n e n t s of the two minor peeks lprobabiy vsrlsnts of the same protelnl a r e sub~ects of a separate study. The materlal recovered from the first step %as subJected to two consecutlve runs on a S1Ze exclusion column (Figs. S . 2 4 . and S.2B.J. The final preparation was lyophilized. Since the as8ay for a n t i p r o l i f e r a t l u e l c y r o t a x l c act~riry 1 3 only e semiquantitative teat, and slnce (as w e demonstrate later in this paper1 our p r o t e ~ n exhiblta RNase activity. w e decided to use the latter for the determination of P-30 Protein recovery durlng the purlflcat~on process. The data are presented in Table S.I.

The final produot was found homogeneous in SDS-PAGE' IFil. s.3.1, as well ss in g e l electrafocuaing (not shown). The equal electrophoretic mabllities of the "on-reduced and reduced ~amples in SDS-PAGE Indicate that it IS a slnqle chain protein. Its noieculsr weight is around l2000, and the isoelectric Point is higher than 9 . 5 .

TUBE NUMBER

ThBLE 5.1.

Recovery of protein and ribonuclease-like activity on

purification of Rana pipiens oocyte anti-tumor protein

Purification step 1mg1 lunitsl ( a ) Activity

~

Extract 635 2 4 9 1 3 3 100

S-Sepharose 4 5 . 4 103053 4 1 . 4

sia-ge1 P-60 (1) 35.9 84280 3 3 . 8

Blo-gel P-60 ( 2 ) 33.5 80166 32.2

Using the presented purification pmcedure we were able to isolate the same protein also from unfertilized Rana pipiens oocytes. The protein prepara- tions Prom the two sources were then Compared ( i n a "heed to head'' manner) usxng ant ipro l i f era t ive l cyrotox ic test, rihonucleoi~t~c actlvlty e a e ~ y , SDS- PAGE. isoelectric focusing and amino acid analysxs (data not shown). They were found indiatinpvlshable in all the tests.

250 Sequence of R. pipiens Oocyte Anti-tumor Protein

1

" -

- kDa - 29.0

21.0

TABLE S.11.

Aa:no a c ~ d com00s1::on o f Ch+ O O C V t e anci-cumor O r O C l i n 0-30 ?ro:e:nl and 1 - 5 cyanaqen bramlde f ~ L Q m e n L D d

ain:no ac:d mOlecOle reoidccs

E n t i r e Ca8r peptides

CNBr-1 cN8c-l- c:IBr-2 1 - 104 1 - 23 2 - 23 24-104

ASP a m 13.9 1:; 6.0 6.0 (61 8 . 5 I81 Thr 9.8b1101 3.1e 3.1e(31 6.7e17) ser 7.8b I81 8.OF(8)

GI" Gl" 6.1 {:! 2.1 Pro

1.1 IiI 4.2 I41

4.1 1 4 ) GlY

4 . 3 I41

ala 3.1 (3) 3.1 I31

cy, 3.0 I31 3.0 (31 8.1' 18) 1.0' 1.1'11) 6.9'171

va 1 xec

8.1 (81 1.2 1.1 (1) 7.i (71 1.0 (11 0.49 0.29(11

I le 5.7d (61 l . g e 1.9e121 3.leI41 Le" 5.1 15) 1.0 1.1 I11 4.1 I 4 1 TyL 3.0 I 3 1 Phe

2.6 I 3 1

H1S 5.9 I 6 1 1.0 3.1 13) 1.1

1.0 I 1 1 5 . 0 151

LYS 12.2 (12) 2.0 1.0 111 2.0 (21 1.9 (21 10.31101

ars 1.0 (1) 1.8 (21

TOL.1 103 104 22.8 21.5(22) 80.7181) ?rp i:: I ? ! i:zh l.Ohlll N.D IO1

Peptide yield Inmoles) 268 86 132

'Residue. per molccu111: values Crom the sequence in parentheses: CN8r-1'. cyanogen bromide fragment 1 with N-terminal pyrogluraaic acid residue removed with pyr0glutamate aminopeptidase: N.O. not

Corrected for destruction: 'as a complex with 3,3'ditiodipr0pionic acid: dCorreCted for incomplete hydrolysis: euncorrected: 'as

bdetetminad.

pyridoethylcysceina: gas homosarine + homoserine laictme: 'estima- ted from KJV-spectrum.

1.9 3.0b

2.1

0.8 1.0

1.1 1.1

2.0

:::e

2.0 (21 J.Ib(31

1.1 Ill

O.Bd(l1 1.0 (11

0.9 I l l 0.9 Ill 1.9 I21 1.0 I11 l.oell)

12.2 I121 6.7'171

4.1 ( 4 1 4.1 ( 4 ) 3.1 (31 3.2 (31

0.9 ( 1 ) 7.9 (81

4.1 ( 4 1 3.8d(51

4.9 I51 2.7 ( 3 1

lO.O(l0) 2.0 (21

2.0 (21 N.0 I O 1

8.Ob181

8.3=(8)

9.6 (101 4.gbl51 5.Sbl61 2.2 (21 2.2 I21 1.2 I 1 1 2.1 12)

4 . 3 I 4 1 0.9 I l l 4.1d151 3.2 0 1 2.0 I 7 1 3 . 1 I 3 1 1.0 I l l 5 . 5 (51 2.0 (21 N . 0 (01

4 . 4 C t 4 )

2.3 (21 2.1'12) 2.3b121

2.2 (21 2.2 I21

1.8 (21

4.2'(41 1.3 I11

3.1 I 4 1

0.8 (I1 1.0 (11

1.9 I21 0.6 (1) 5.1 (51

N.0 I O 1

3 . 3 (31 3.Ob0I 3.4b(31 1.2 I 1 1 1.1 I11

4 . 1 C I 4 1 1.9 (21

1.9 (2) 1.4 (21 1.6 I21

4.9 ( 5 1 1.0 I11 N.0 I O 1

1.1 (11

1.1 I l l 1.0 11)

2.0 I21 1.1 (11

1.0 ( 1 1

3.0 ( 3 ) 0.8=(11

0.9 (11 1.0 ( 1 1

N . 0 101

Total 15.1 13.71141 88.0(891 57.91581 30.91311 28.8(29) 13.01131

I'rpLide yicld Inmoles1 280 95 97 71 74 122 108

'Residues per molecule: T-1 to T-4, tryptic peptides generated from a CitraCOnylated P-30 derivative: T-I., tryptic Craglnent 1 with the N-terminal pyroglutamic acid residue removed by pycoglutanate aminopeptidase: SP-3 and SP-4, peptides generated with StaphyloCOCCaI

Uncorrected Cor destruction: 'as pyridoethyleysteine: dUnCOrreCted for inconplete hydrolysis ,,proteinase v 8 .

'estimated Crorn Uv-spectrum.

TABLE S . I V .

CNBr-lS(2.1) CNBI"214.7) Cycle Cycle Cycle

1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 17 18 19 20 21 22 23 23

5 773 T 663 N 2259 L 2422 F 2128

1053 :a 2369 K 1881 0 1685 K 2195 N 1778 T 533 F 1342

V 1054 I 916

5 179 R 775

E 571 P 682

V 326 ? 487

R 522 A 309

24 25 26 27 28

30 29

31 32 3 3 34 35 36 37 38 39 40 41 42 43 44 4 5 46

358 576 439 231 229 275 252 39 269 187 141 150

6 1 79 16 35 77 67 53 12 41

141 83

s.qu.nc. position 2-23 24-104

Sequence of R. pipiens Oocyte Anti-tumor Protein

I I TABLE S . V I .

251

B L Z . 5.4. Seoararlon of the t ~ y ~ t i c disest of R.o~plens oooyte anti-tumor

Reduced, pyridoethylated. and oltraconylated derlvarlve of the pmtein w a s digested. as deacrlbed under Methods. The dkgerrt "ea chromatographed on B

2.5 x 4 7 Cm Bio-Cel P-60 column in 611 z u m i d i n e hydrochloride. 0 . 5 X sodium chloride, pH 2.0. Three nl fractions were collected at the flow rate of 1 1 ml/h. Peptides wepe numbered in order of their appearance in the sequence. Solid bars indlcate the fractions combined.

orotein.

I\

Sequence analysis of peptides qenerated from reduced and alkylated P-30 Protein with 5 . a Y r e Y S V 8 ProLelnaSe

Partial sequence of the SP-2 peptide was determined from its mixture with the N-terminal (blocked1 fragment: the amount used tor the run is, therefore, not xeported. The arrow indicates C-terminal residue

of the peptxde. Other details 19 in Table 5.111.

8 9 10

E 2742 Y 2499 L 2114 s 735 D 1976 Ca 2289 N 2105 Y 1882 T 511 5 436

11 R 1256 12 P 1356 13 Ca 2279 - 15 Y 1323 I1 K 3351

16 K 2774 17 L 1090 18 K 2475

N 6981 Q 9672 A 9247 P 6755 V 3628 H 2147 F 3387 Y 2244 C 1301 V 1343

Y 3273 F 4567

L 5982 5 1060 D 1868 Ca 1931b N 440 V 2442 T 1113 s 335

C 1365 R 1372 5 141

X I010 Y 390 K 698

K 571 L 547

~ . .

Sequence Position 63-104 92-104 63-91

height of nineteen PTH-amino acid standards. a A S pyridoethylcysreine: 'the yield was extimated using the mean peak

1 D 1582 2 W 243 3 L 1025 4 T 400 5 F 579 G 0 195 7 K 104 0 K 193

IO I 212 9 tI 117

I1 T 60

13 T 37 I? N 82

;;+R 33 16 I7 18 I? 7"

D 3148 V 4231

3 3 N 2MIl D 2169

I 2513 E( 2287 5 494 T 639 N 1383

F 1367 L 2040

3 1 3 K 1693 0 1379 K 1550 N 712 T 468

D 3064 24 5 86 V 3560 25 R 811 D 4425 26 P 385 Ca 2690' 27 E 169 D 4250 28 P 273 N 3073 29 Y 159 I 2386 30 K 168 M 2697 31 R 140 S 984 32 I 154< T 1059 33 ca --- N 853 34 I( 133

F 1331 36 1 134 L 1047 .35 c 47

H 1149 37 1 181 Ca 1143b 38 A 78 K 1277 39 5 I4 D 1224 40 K 72 K 1200 41 N 40 N 756 42 Y 55 I 308' 43 L 52

19 20 21 22 23 24 25 26 27 28

K 440

T 53 5 31

N 59 K 136 ,'a izb

V 63

T-l'(3.71 T-217.8) T-317.41 T-4(16.I)

Cycle Cycle Cycle Cycle

".,~ ~~

21 F 752 44 T 32 22 I 805 45 T 9 23 Y 472

10 11 12 13 11 15 16 17 18 19 20

P 11024 21 A 5034 C* 12781 22 P 3301 K ---C 23 V 3202 Y 83W 24 H 1301 K 12013 25 F 2567

K 10515 27 G 2094 L 8855 26 V 2588

R 9658 28 V ---' S 2187 29 C 2295 T 3655 30 5 301 N 6013 31-C 875 Y 7,m .. F 6393 Ca 9072 V 7217 T 16% C 7287 E 4753

0 3264 N 5348