J. Biochem. 111, 157-161 (1992)

Primary Structures of Sardaines Z1 and Z2, Protamines Isolated from St riped ( orientalis)

Yoshiko Okamoto, * Katsutoshi Kuno , * * Terushige Motohiro, * * Norio Nishi, * * * Eiko Muta, * and Shoshi Ota* *Department of Biochemist **Laboratory of F ry, Daiichi College of Pharmaceutical Sciences, Minami-ku, Fukuoka, Fukuoka 815; K ood Quality Control and Analysis, Faculty of Fisheries , Kagoshima University, Kagoshima, H agoshima 890; and -Department of Polymer Science, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, okkaido 060

Received for publication, October 8, 1991

Striped bonito protamine, sardaine, was isolated from the sperm of striped bonito (Sarda orientalis) by extraction with sulfuric acid followed by ion-exchange chromatography. The preparation gave a single band upon polyacrylamide gel electrophoresis. Sardaine consists of 34 amino acid residues, and its sequence is: Pro-Arg-Arg-Arg-Arg-Arg Ser(Ala)-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg-Tyr-Arg-Arg-Ser-Thr-Ala-Ala .-Arg-Arg-Arg-Arg Arg-Val-Val-Arg-Arg-Arg-Arg. At position 7, serine (sardaine Z1) is partially replaced by alanine (sardaine Z2). The ion spray mass spectrum shows that sardaines Z1 and Z2 have molecular masses of 4,612.49 and 4,596.09 Da, respectively. The sequence of sardaine Z1 is 100% identical with that of thynnine Z2 from tuna fish (both fish belong to , ).

Protamines are low-molecular-weight, highly basic pro boxypeptidase A (CPA) [EC 3.4.12.1], and DFP-treated teins which are found in the spermatic cells of fish (1-3), carboxypeptidase B (CPB) [EC 3.4.12.3] were obtained birds (4-6), and mammals (7-19). The newly synthesized from Sigma Chemical, St. Louis, U.S.A. protamines are phosphorylated on serine residues. The Purification of Sardaine-The extraction was conducted phosphorylated protamines are transferred into chromatin, according to Honma (23). The proteins were extracted where they replace the histones and bind to DNA to form from minced and homogenized milts by stirring in 5% nucleoprotamine. During the maturation of the sperm, the sulfuric acid for 2 h followed by centrifugation. The extrac protamine is dephosphorylated and the nucleoprotamine tion procedures were carried out repeatedly. Two volumes becomes more compact (20-22). In this compact structure, of ethanol were added to the collected extract, and the transcription is entirely inhibited and DNA is protected whole was allowed to stand overnight at O'C. The precipi from enzymatic hydrolysis. Our knowledge of the mode of tate was obtained by centrifugation at 4,000 x g for 20 min, packing and organization of DNA is still fragmentary, and and was dissolved in hot water. Insoluble materials were so, to gain a better understanding of protamine-DNA removed by centrifugation at 15,000 x g for 20 min, then interaction, it is valuable to identify the sequences of the supernatant was allowed to stand overnight once again various protamines. In the present studies, sardaine iso at O'C. The precipitate was redissolved in a small volume of lated from the milts of striped bonito (Sarda orientalis) hot water and lyophilized (Extract-I). The pH of Extract-I was purified as a single component by ion-exchange chro dissolved in distilled water was adjusted to pH 10.5. The matography, and the amino acid sequence and the molecu solution was allowed to stand overnight at 0'C, and then was lar mass were determined. The sequence of sardaine was centrifuged at 30,000 x g for 20 min, to remove the precipi then compared with the sequences of tuna fish and For tate. Proteins were precipitated by adding 2 volumes of mosan grey mullet protamines. ethanol and a few drops of diluted sulfuric acid and allowed to stand overnight at O'C. The precipitate was dissolved in MATERIALSAND METHODS a small volume of distilled water and lyophilized (Extract II). Starting Materials-Striped bonito were collected from Extract-II (200 mg) was dissolved in 0.1 M sodium the waters around Yakushima and Tanegashima islands, in acetate buffer (pH 6.0) containing 1.6 M NaCI and then was the southern part of Japan from May to June, 1985. The applied to a CM-Sephadex C-25 (Pharmacia Fine Chemi milts were removed from the freshly caught fish and then cals) column (2.5 x 80 cm) which had been equilibrated frozen immediately at 20'C. with the same buffer. Protamine was eluted by the same Reagents-All chemicals were of the best grade commer buffer. Protamine concentration of the fraction was deter cially available. Pepsin [EC 3.4.23.1], DFP-treated car mined by measuring either the absorbance at 230 nm, or the absorbance at 500 nm after the Sakaguchi reaction. The protamine-containing fractions were pooled and desalted Abbreviations: DFP, diisopropylfluorophosphate; CPA, carboxypep on columns of CM-Sephadex C-25 (3 x 15 cm) and Amber tidase A; CPB, carboxypeptidase B; PTH, phenylthiohydantoin; lite IRA-47 (2 x 25 cm). Once again the protamine DTT, dithiothreitol; FAB-MS, fast atom bombardment mass spec trometry; IS-MS, ion spray mass spectrometry. containing fractions were pooled and adjusted to pH 5.0

Vol. 111, No. 2, 1992 157 158 Y. Okamoto et al. with 0.5 N sulfuric acid to form protamine sulfate, and then liquid chromatograph (28, 29). lyophilized (CM-I). The sample (500 pmol) was dissolved in water (100 CM-I (100 mg) was dissolved in 0.1 M sodium acetate pmol/pl), and was separated by HPLC on a Develosil buffer (pH 6.0) containing 1.0 M NaCI and then was applied 3000DS-5 column (1.5 X 150 mm, Nomura Chemicals). to a CM-Sephadex C-25 column (2.5 X 80 cm) which had The elution was performed with a 0-50% linear gradient of been equilibrated with the same buffer. After washing of acetonitrile in 0.05% trifluoroacetic acid for 25 min, at a the column with 100 ml of the same buffer, elution was flow rate of 0.1 ml/min. One-sixteenth of the eluate was performed with a linear gradient (2 x 500 ml) of 1.0-2.5 M passed to the ion source. The ion spray ion source was NaCl in 0.1 M sodium acetate buffer (pH 6.0). After operated at 4,500 V with 35 psi nebulizing gas (compressed desalting, protamine was lyophilized as the sulfate (sar air) at a flow rate of 0.8 liter/min, and the interface plate daine). and the orifice voltages were adjusted at 650 and 90 V, Pepsin Digestion of Sardaine-Sardaine (18.5 mg) dis respectively. During the on-line chromatographic analysis, solved in 0.1 M sodium acetate buffer (pH 4.0) containing the mass spectrometer was scanned from m/z=380 to 800 2.0 M NaCI was digested with pepsin at 37TC (substrate: or m/z=400 to 2,000. Mass calibration was performed enzyme= 200: 1, w/w) (24). After incubation for 2 h, the using charged ions from 0.1 mM polypropyleneglycol 1,000 reaction was terminated by keeping the preparation at and 2,000 dissolved in acetonitrile/1 mM ammonium 100°C for 3 min. The preparation was diluted with distilled acetate solution (4: 1, by volume). The molecular masses water, and then applied to a CM-Sephadex C-25 column of the peptides were calculated using the automated (2.5 x 80 cm). Fractionation and desalting were carried out Hypermass computer program, which is based on the according to the procedures described above. following equation: Gel Electrophoresis-Sardaine and the digestion prod ucts were analyzed according to Reisfeld et al. (25). The gel was polymerized in a glass dish (0.75 x 7 x 8 cm), using 20 ml of small-pore solution and 8 ml of large-pore solution. Then 2 g of the sample was dissolved in 2 ,u1 of 30% sucrose and was charged on each groove. A constant current where m, and m2 are mass-to-charge ratios of any adjacent of 28 mA/slab was applied to the anode in the upper peaks, n is the charge state, H is the mass of a proton, and chamber for 1.25 h at room temperature using a Mini M is the molecular weight. protean II (Bio-Rad). The gel was stained with 0.114% Coomassie Brilliant Blue R-250 in 5% formic acid and 2.7% RESULTS methanol for 40 min, and then destained with 3.5% formal dehyde in 24.8% methanol. Purification-The extraction of protein with 5% sulfuric Amino Acid Analysis-Samples were hydrolyzed in acid from striped bonito milts yielded protamine in an vacuo at 110°C for 24h in 6 N HCI. All analyses were almost pure state. Protamine was eluted from a CM performed according to the methods of Spackman et al. Sephadex C-25 column by isocratic elution as a single (26) on a JEOL JLC-200A amino acid analyzer. component. When herring, salmon and tuna fish protamines Automated Sequence Analysis-Automated Edman deg (30, 31) were chromatographed under similar conditions, radation of proteins (2.0 mg) was performed in a JEOL they were each resolved into several components. The JAS-570K sequence analyzer, using a peptide program. chromatography of the protamine of striped bonito on Prior to sequencing, the program was run for two cycles CM-Sephadex C-25 with linear gradient elution resulted in with 2 mg of polybrene. A program with double coupling a single peak, as in the case of pike protamine (32) (Fig. 1). and double cleavage was used on the first cycle and for the proline residue. The resulting PTH-derivatives of amino acids were dissolved in methanol which contained 0.05 mM norleucine as an internal standard, and then analyzed by HPLC on a column of Novapak C18 (Waters). PTH derivatives were separated by a gradient of 2-propanol and 0.02 M acetate buffer (pH 4.98) containing 17% aceto nitrile. Carboxy Terminal Analysis-Sardaine (3.0 mg) dis solved in 0.5 ml of 0.02 M Tris-HCI buffer (pH 7.7) was digested at 37°C with DFP-treated CPB (substrate:en zyme=750:1, mol/mol) (27). After 4h, the reaction mixture was kept at 100°C for 5 min. DFP-treated CPA was then added to the remainder of the preparation (substrate: enzyme = 500:1, mol/mol). The time course of the carboxy peptidase digestion was followed by analyzing 0.02 ml aliquots of the reaction mixture at intervals, using an amino acid analyzer after the enzyme reaction had been stopped by the addition of 0.2 M citrate buffer (pH 2.2). Fig. 1. CM-Sephadex C-25 column chromatography of sar Ion Spray Mass Spectrometry-Ion spray mass spec daine. Details are given in "MATERIALS AND METHODS." The trometry (IS-MS) was performed on a Perkin Elmer Sciex flow rate was 20 ml/h, and fractions of 10 ml were collected. The API-III quadrupole mass spectrometer coupled with a eluted fractions were monitored at 230 nm.

J. Biochem. Primary Structure of Sardaine 159

Fig. 2. Polyacrylamide gel electropho resis of sardaine and other protamines . Electrophoresis was carried out at 28 mA for 1.25 h. Migration was from top (anode) to bottom. 1, clupeine; 2, salmine; 3, mugiline,6 M6; 4, sardaine.

TABLE I. Amino acid compositions of sardaine and its pepsin digests (P-I, P-II). The results are expressed as residues/molecule . The numbers in parentheses represent the composition from sequence analysis.

'Values for threonine and serine are zero -hydrolysis-time extrapola tions.

When salmon (32) and mullet protamines (3) were chro matographed under similar conditions they were each resolved into several peaks of proteins. Sardaine thus Fig. 3. Amounts of PTH-amino acids from automated Edman prepared migrated as a single band upon polyacrylamide degradation of sardaine. A 414 nmol sample of sardaine was used for automated sequencing and PTH-amino acids were identified by gel electrophoresis according to Reisfeld et al. (25) (Fig. 2) and Spiker (33) (data not shown). It migrated more slowly HPLC. PTH-Thr was identified as PTH-dehydrothreonine. PTH-Ser was identified as PTH-dehydroserine DTT-adduct and expressed as a than herring, salmon, and mullet protamines. percentage of the amount of internal standard (norleucine) recovered. Amino Acid Composition-The amino acid composition PTH-Val and PTH-Tyr are indicated by dotted lines in the respective of sardaine is presented in Table I. Only seven types of plots. amino acids could be detected. Arginine accounts for about two-thirds of the total mol%. Tyrosine has rarely been found in other fish protamines. Automated Protein Sequencing-The results of automat ed Edman degradation on sardaine are shown in Figs. 3 and 4. The sequence of the C-terminal region was confirmed by CPB and CPA digestion: 3.70 mol of arginine, followed by 1.71 mol of valine were released per mol of sardaine (Fig. 5). Sequence analysis showed that serine and alanine were detected simultaneously at position 7 (Fig. 3). This is the reason why the numbers of residues of serine and alanine based on 34 residues per molecule were not near-integers (Table I). Pepsin Digestion-Sardaine was digested with pepsin, and the products were separated on CM-Sephadex C-25. Two fragments (P-I and P-II) were found (Fig. 6), and each migrated as a single band on 15% polyacrylamide gel Fig. 4. Amino acid sequences of sardaines Z1 and Z2. The heterogeneity in position 7 is indicated by assigning both serine and electrophoresis (Fig. 7). As shown in Table I, the amino acid alanine. (-h), Automated Edman degradation; (- ), CPB and CPA composition of sardaine was in good agreement with the digestion. sum of P-I and P-Il. The sequencings of P-I and P-II confirmed the sequence of the intact sardaine (Fig. 4). Mass Spectrometry of Sardaine-From the amino acid composition and the sequence analysis, it was suggested subjected to mass spectrometric analysis. Sardaine was that sardaine contained two slightly different components first submitted to fast atom bombardment mass spectro metry (FAB-MS). A positive result, however, could not be (Z1, Z2). In order to verify these results, sardaine was

Vol. 111, No. 2, 1992 160 Y. Okamoto et al.

Fig. 7. Polyacrylamide gel electropho resis of pepsin digests of sardaine. Electrophoresis was carried out at 28 mA for 1.25 h. 1, sardaine; 2, pepsin digests of sardaine; 3, P-I; 4, P-II.

Fig. 5. Hydrolysis of sardaine with CPB and CPA. Sardaine was digested with CPB at 37°C for 4 h, followed by CPA for 4 h. Sardaine: CPB= 750:1, sardaine:CPA=500:1 (mol/mol).

Fig. 8. Ion spray mass spectrum of sardaine. Sardaine (500 pmol/5 pl) dissolved in water was applied. Two series (A) and (B) of multiply charged ions with 7 to 11 charges were detected.

Fig. 6. CM-Sephadex C-25 column chromatography of pepsin digests of sardaine. Details are given in "MATERIALS AND METHODS." The flow rate was 20 ml/h, and fractions of 10 ml were either display or separate protamine components from collected. The eluted fractions were monitored at 230 nm. other types of fish. The heterogeneity at position 7 of sardaine was considered to be due to a partial substitution of serine for alanine caused by one base change. No example obtained, probably because of its very high arginine con is known of protamines which have the same amino acid tent. When sardaine sulfate was subjected to IS-MS sequence from different fish species, even if the species analysis, many peaks which could not be assigned were belong to the same order. However, the sequence found observed. Finally a clear spectrum was obtained from here, which has serine at position 7 of sardaine from striped desulfated sardaine by means of LC/MS. As shown in Fig. bonito (sardaine Z1), is exactly the same as the sequence of 8, IS-MS confirms the existence of sardaines Z1 and Z2, thynnine Z2 from tuna fish (34) (Fig. 9). Both fish belong to since two series (A, B) of multiply charged ions are visible the same suborder, Scombridae. In Fig. 9, sardaine is in the spectrum. The molecular mass measured for series aligned with mugiline /3 and thynnine to emphasize the A was 4,612.49±0.74 Da, which corresponded to the arginine clusters and sequence homology among them. monoisotropic mass (4,610.83 Da) and the average mass Mullet, tuna fish, and striped bonito belong to the same (4,613.46 Da) of sardaine Z1. The molecular mass mea order, Perciformes. sured for series B was 4,596.09±1.10 Da, and also corre Serine residues in protamine are phosphorylated during sponded to the monoisotropic mass (4,594.83 Da) and the the transfer from the cytoplasm to the nucleus and are average mass (4,597.46 Da) of sardaine Z2. dephosphorylated on the DNA with the replacement of the histones (20-22). The role of phosphorylation of serine DISCUSSION seems to be to facilitate the transfer of the highly positively charged protamine molecules into the nucleus. Since Ion-exchange chromatography and gel electrophoresis protamines seem to be synthesized in order of increasing produced no evidence for variations in either size or charge basicity and decreasing serine/phosphate content, sardaine of sardaine, though both techniques have been used to Z2 (alanine at position 7) may be produced at a later stage

J. Biochem. Primary Structure of Sardaine 161

Fig. 9. Comparison of the se quences of fish protamines be longing to Perciformes. Regions of arginine clusters are boxed: mugiline /3, Mugil japonicus; thyn nine, Thynnus thynnus; sardaine, Sarda orientalis.

than sardaine Z1 during spermatogenesis. J. Biochem. 144,121-125 Proline is generally present at two or three residues per 12. Tobita, T., Tsutsumi, H., Kato, A., Suzuki, H., Nomoto, M., molecule in fish protamines. The absence of the third Nakano, M., & Ando, T. (1983) Biochim. Biophys. Acta 744,141 proline in sardaine should cause it to have a less compact 146 conformation as compared to those of herring, salmon, and 13. Mazrimas, J.A., Corzett, M., Campos, C., & Balhorn, R. (1986) Biochim. Biophys. Acta 872, 11-15 mullet protamines, which contain three prolines. There 14. Ammer, H. & Henschen, A. (1987) Biol. Chem. Hoppe-Seyler fore, it seems reasonable that sardaine shows a slower 368,1619-1626 mobility upon gel electrophoresis in spite of their similar 15. Belaiche, D., Lair, M., Kruggle, W., & Sautiere, P. (1987) molecular sizes and charge densities. Biochim. Biophys. Acta 913, 145-149 It has been reported that tyrosine is contained in the 16. Pirhonen, A., Valtonen, P., Linnala-Kankkunen, A., Heiskanen, M.-L., & Maenpaa, H. (1990) Biochim. Biophys. Acta 1039,177 internal position of some protamines such as four compo 180 nents of thynnine and scylliorhinine Z3. In addition, 17. McKay, D.J., Renaux, B.S., & Dixon, G.H. (1985) Biosci. Rep. 5, tyrosine in scylliorhinine Z3 is modified in its phenolic 383-391 group with the formation of tyrosine 0-sulfate (35). In the 18. McKay, D.J., Renaux, B.S., & Dixon, G.H. (1986) Eur. J. present study, sardaine was also found to contain tyrosine Biochem. 156, 5-8 at position 17, but no evidence was found for its modifi 19. Ammer, H., Henschen, A., & Lee, C.-H. (1986) Biol. Chem. Hoppe-Seyler 367, 515 522 cation. 20. Ingles, C.J. & Dixon, G.H. (1967) Proc. Natl. Acad. Sci. U.S.A. 58,1011-1018 The authors are indebted to Yasutsugu Shimonishi and Toshifumi 21. Marushige, K., Ling, V., & Dixon, G.H. (1969) J Biol. Chem. Takao (Institute for Protein Research, Osaka University) for FAB 244,5953-5958 MS analysis, and Yoshihisa Umeda (Biotechnology Research Labora 22. Louie, A.J. &Dixon, G.H. (1974) Can. J. Biochem. 52, 536-546 tories, Takara Shuzou Co.) for IS-MS analysis. We also thank Brian 23. Honma, T. (1960) Bull. Jpn. Soc. Sci. Fish. 26, 21-24 T. Quinn for critical comments on our manuscript. 24. Bretzel, G. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 933 943 REFERENCES 25. Reisfeld, R.A., Lewis, U.J., & Williams, D.E. (1962) Nature 195,281-283 1. McKay, D.J., Renaux, B.S., & Dixon, G.H. (1986) Eur. J. 26. Spackman, D.H., Stein, W.H., & Moore, S. (1958) Anal. Biochem. 158, 361-366 Biochem. 30, 1190-1206 2. Chevaillier, P., Martinage, A., Gusse, M., & Sautiere, P. (1987) 27. Nakano, M., Tobita, T., & Ando, T. (1973) Int. J. Pept. Protein Biochim. Biophys. Acta 914, 19-27 Res. 5,149-159 3. Okamoto, Y., Muta, E., & Ota, S. (1987) J. Biochem. 101, 1017 28. Bruins, A.P., Covey, T.R., & Henion, J.D. (1987) Anal. Chem. 1024 59,2642-2646 4. Oliva, R., Goren, R., & Dixon, G.H. (1989) J. Biol. Chem. 264, 29. Covey, T.R., Bonner, R.F., Shushan, B.I., & Henion, J.D. (1988) 17627-17630 Rapid Commun. Mass Spectrom. 2, 249-256 5. Nakano, M., Tobita, T., & Ando, T. (1976) Int. J. Pept. Protein 30. Ando, T. & Watanabe, S. (1969) Int. J. Pept. Protein Res. 1, 221 Res. 8, 565 578 224 6. Oliva, R. & Dixon, D.H. (1989) J. Biol. Chem. 264,12472-12481 31. Breyzel, G. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352,1025 7. Kleene, K.C., Distal, R.J., & Hecht, N.B. (1985) Biochemistry 1033 24,719-722 32. Speckert, W., Kennedy, B., Daisley, S.L., & Davies, P. (1983) 8. Yelick, P.C., Balhorn, R., Johnson, P.A., Corzett, M., Mazrimas, Eur. J. Biochem. 136, 283-289 J.A., Kleene, K.C., & Hecht, N.B, (1987) Mol. Cell. Biol. 7, 33. Spiker, S. (1980) Anal. Biochem. 108, 263-265 2173-2179 34. Bretzel, G. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 543 9. Ammer, H. & Henschen, A. (1988) Biol. Chem. Hoppe-Seyler 549 369,1301-1306 35. Sautiere, P., Briand, G., Gusse, M., & Chevaillier, P. (1981) Eur. 10. Ammer, H. & Henschen, A. (1988) FEBS Lett. 242, 111-116 J. Biochem. 119, 251-255 11. Sautiere, P., Belaiche, D., Martinage, A., & Loir, M. (1984) Eur.

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