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Vol. 189, No. 3, 1992 December 30, 1992

CALCYCLIN

AND CALVASCULIN

EXIST

IN HUMAN

PLATELETS

Yasuhiro Tomida $, Motomu Terasawa,Ryoji Kobayashi and Hiroyoshi Hidakat Department of Pharmacology and $Thoracic Surgery, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan Received

November

7,

1992

Summary: Using Ca 2+-dependentaffinity chromatography on a synthetic compound (W-7)-coupled Sepharose column, three distinct Ca2+-binding proteins have been identified in human platelets. The molecular mass of these three distinct proteins was estimated to be 10, 10.5, 17 kDa,respectively, by polyacrylamide gel electrophoresis in the presence of SDS. The partial amino acid sequencerevealed these proteins have EFhand structures and high homology to the predicted proteins, calcyclin, calvasculin, and calmodulin. Calcyclin and calvasculin have been considered as probably having roles in the control of cell proliferation, but the existence of these two proteins in platelets suggests that they have other intracellular functions related to the Ca2+-signal transduction system. 0 1992Academic Pre.ss,Inc.

Human platelets are one of the tissues available for biochemical experiments and the smallest of the many varieties of cells in circulating blood. Despite their small size, platelets play important roles in defense reaction following vascular injury and in blood coagulation in hemostasis. Platelets respond to a variety of physiological and nonphysiological

stimuli by undergoing complex biochemical and morphological

changes. Platelet functions such as adhesion, aggregation and secretion are tentatively explained to be mainly regulated by Ca2+-calmodulin (CaM) dependent phosphorylation of myosin light chain catalyzed by myosin light chain kinase (l-3). But the existence of an intricate network in signal transduction, that is made up of interacting systems regulated not only by CaM but also by protein kinase C (4), tyrosin kinase (5) ~To whom correspondence should be addressed. The abbreviations used are: EGTA, ethylenebis (oxyethylenenitrilo) tetraacetic acid; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol; W-7, (N-(6-aminohexyl)-5-chlorol-naphthalenesulfonamide); kDa, kilodalton( SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and protein kinase A (6), has been suggested with the progress in studying Ca*+-signal transduction system studies in platelets. CaM antagonists, for example chlorpromazine, trifuluoperazine,

W-7 (N-(6-aminohexyl)-5-chloro-

1-naphthalenesulfonamide), have

often been used for elucidating the physiological functions of CaM (7). Furthermore, CaM antagonists are proven to be good affinity ligands to purify CaM and other Ca*+binding proteins (g-10). In this study, using W-7 affinity chromatography, we report the purification and partial sequence analysis of 10 and 10.5 kDa Ca*+-binding proteins in human platelets.

MATERIALS

AND

METHODS

Reagents: All chemicals were of reagent grade. Cyanogen bromide-activated Sepharose 4B and Q-Sepharose were purchased from Pharmacia-LKB. W-7 was synthesized according to the method of Hidaka ECal (11). Nitrocellulose filter paper was obtained from Millipore Co. Horseradish peroxidase-conjugated anti-rabbit IgG (Fab’ fragment) was obtained from MBL. Lysylendopeptidase was obtained from Sigma. Preparation of a W-7 Sepharose Affinity Column: The coupling of W-7 to cyanogen bromide-activated Sepharose 4B was carried out as described by Endo et al (8). Protein purification: Platelet-rich-plasma (PRP) was prepared from titrated whole blood from healthy donors. PRP was mixed with 4 volumes of buffer A, containing 0.2 mM EGTA, 40 mM Tris-HCl (pH 7.5), and centrifuged for 10 min at 700 x g at room temperature and the supernatant was wasted. This operation was repeated three times, and the washed platelets, resuspended with 4 volumes of buffer A, were homogenized by a ultrasonic disrupter (TOMY, UD-201). The homogenate was centrifuged at 100,000 x g for 60 min at 4 “C. CaC12 and NaCl were added to the supernatant at a final concentration of 2 mM and 0.5 M, respectively. The solution was centrifuged at 100,000 x g for 20 min at 4 “C and then the supematant was applied to the W-7 Sepharose 4B column preequilibrated with buffer B: 0.5M NaCl, 2 mM CaClz, 40 mM Tris-HCl (pH 7.5). Further purification procedures are described in the Results section. Analytical Procedures Electrophoresis: SDS-PAGE was performed according to the method previously reported (12). After electrophoresis, the gels were stained with Coomassie Brilliant Blue. Partial amino acid sequence: Enzymatic digestion by lysylendopeptidase was done for the sample of 10 or 10.5 kDa protein with buffer C, containing 40 mM Tris-HCl (pH 7.5), at 37°C for an appropriate time. The resulting peptides were separated by reverse-phase chromatography on a cl8 column (STR ODS-M 4 x 150 mm) on a Shimadzu HPLC system (LC4A), which were monitored by their absorbance at 215 nm, with a O-80% acetonitrile linear gradient in the presence of 0.1% trifluoroacetic acid for 80 min at a flow rate of 1 ml/min. The amino acid sequence determination for each proteolytic fragment was performed on an Applied Biosystems (Foster City, CA) 473A pulse-liquid sequenator equipped with an on-line analyzer for the phenylthiohydantoin derivatives.

RESULTS Protein

Purification:

The supernatant prepared from platelets was applied to

the W-7 Sepharose 4B column which was preequilibrated with buffer B. The column 1311

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SDS-PAGE

10

Idion

15

20

Number (8 ml/tube)

Fig. 1. W-7Sepharose affinity chromatography of human platelet Ca*+-binding proteins. EGTA extract from human platelets was applied to a W-7-Sepharose affinity column (3 x 15 cm) preequilibrated with 0.5 M NaCl, 2 mM CaC12,and 40 mM TrisHCl (pH 7.5).The column was extensively washed with the same buffer and eluted with 0.5 M NaCl, 5 mM EGTA, and 40 mM Tris-HCI (pH 7.5). Fractions of 10 ml were collected. The inset shows SDS-PAGE run with 30 pl of the 8th fraction proteins.

was washed with the same buffer until the buffer was protein free. Then calcium binding proteins were eluted with 0.5 M NaCl, 5 mM EGTA, 40 mM Tris-HCl (pH 7.5). The elution profile revealed a single protein peak (Fig. 1) and SDS-PAGE showed three major bands and several minor bands. These minor bands were identified as annexin-family proteins with anti-annexin family protein antibodies (unpublished results). The EGTA-eluate was required to separate the 10 and 10.5 kDa proteins from 17 kDa protein (apparently CaM). Protein rich fractions were pooled and dialyzed extensively against buffer C, and then applied to a Q-Sepharose column preequilibrated with the same buffer. The column was washed with 3 volumes of the same buffer and eluted with stepped gradients of 0, 0.2, 0.5 M of NaCl in buffer C. The elution profile indicated two peaks (Fig. 2), and SDS-PAGE showed two proteins in prior fraction and 17 kDa protein afterward. Finally, the two proteins were separated by reverse-phase liquid chromatography on a Cl8 column on a Shimadzu HPLC system with the same way described in a part of “partial amino acid sequence.”The chart of HPLC showed two peaks (Fig. 3), and SDS-PAGE revealed a distinct single band of 10 or 10.5 kDa in the respective peak.

Partial Amino Acid Sequence Analysis: Amino acid sequenceanalysis was performed as described above. A computer homology search of these sequences revealed a high homology to calcyclin and calvasculin for 10 and 10.5 kDa protein, 1312

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Fraction

20

25

30

A

0

B

35

Number (4 ml/tube)

Fig. 2. Separation of human platelet Ca 2+-binding proteins by Q-Sepharose chromatography. Pooled, dialyzed, protein-rich fractions from the W-7-Sepharose affinity column were applied to a Q-Sepharose column (2 x 10 cm) preequilibrated with 40 m M Tris-HCl (pH 7.5). The column was washed with the same buffer and then eluted with a stepped gradient of 0, 0.2, 0.5 M of NaCl in 40 m M Tris-HCl (pH 7.5). Fractions of 4 ml were collected. The inset shows SDS-PAGE analyses of selected fractions obtained from the Q-Sepharose column. A, SDS-PAGE run with 30 pl of the 12th fraction proteins. Two proteins of molecular weights 10 and 10.5 kDa were found in the gel stained with Coomassie Brilliant Blue R-250. B, SDS-PAGE with 30 p.l of the 25th fraction protein.

respectively.Fig. 4 provides comparisonsof the partial amino acid sequencesof human platelet 10 kDa protein with the corresponding sequencesof rat, rabbit and human calcyclin, those of human platelet 10.5 kDa protein with the sequencesof bovine

.

SDS-PAGE

-

A-l

A-2

II

A-i A-2 Retention Time

Fig. 3. Separation of human platelet Ca2+-binding proteins on C I 8 reversephase column chromatography. Pooled, protein-rich fractions from the Q-Sepharose column were applied to a C1s reverse-phase column (STR ODS-M 4 x 150 mm) preequilibrated with 0.1% trifluoroacetic acid and eluted with O-80% acetonitrile linear gradient in the presence of 0.1% trifluoroacetic acid for 80 min at a flow rate of 1 ml/min. The inset shows SDS-PAGE run with 30 p1 of the respective fraction protein. 1313

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A. Calcyclin 1 Human platelet Rat 2A9 Rabbit

11

MACPLDQAIGLLVAIFHKYSGREGDKHTLSKKELKELIQKELTIGSKLQD EGDKHGLSK

*

50 LQD

*

ELIQKELGIGSK El*

~EIARLMEDL~RNKDQEVNF~EYVTFLGALALIYNEAXX AEIARLMDDLDRNKDQEVNFQEYATFLGALALIYNEALKG AEIARLMEDLDRNKDQEVNFQEYVTFLGALALIYNEALKG LMDDLDRNKDQEVNFQEYITFLGALAVIYNEALK

* 11

1

* 21

*

*

6,

*

*

71

*

31

ALDVMVSTFHK ALDVMVSTFHK ARPLEEALDVI VSTFHKYSGKEGDKFKLNKTELKELLTRELPSFLGKRTD ARPLEEALDVIVSTFHKYSGNEGDKFKLNKTELKELLTRELPSFLFKRTD ARPLEEALDVI VSTFHKYSGKEGDKFKLNKTELKELLTRELPSFLGRRTD ARPLEEALDVI VSTFHKYSGNEGDKFKLNKTELKELLTRELPSFLGRRTD

St*

Human platelet Bovine aorta pEL-98 18A2 42A p9Ka

41 *

MACPLDQAIGLLVAIFHKYSGREGDKHTLSKKELKELIQKELTIGAKLQD

9. Calvasculin Human platelet Bovine aorta pEL-98 18A2 42A p9Ka

31

COMMUNICATIONS

HTLSK

*

Humanplatelet Rat 2A9 Rabbit

*

21

RESEARCH

ELLTRETPSFLGKRT: ELLTRELPSFLGKXTD

81

91

100

EA A F Q K ETAFQKLMSNLDYNKDNEVDFQEYXVFL EAAFQKVMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGCPDKEPRKK EAAFQKVMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGCPDKEPRKK EAAFQKLMNNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGCPDKEPRKK EAAFQKLMNNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGCPDKEPRKK

Fig. 4. The comparisons of the partial amino acid sequences of rat, human fibroblasts (2A9), rabbit and human platelet calcyclin, and of pEL-98, 18A2, 42A, p9Ka, bovine aorta calvasculin and human platelet calvasculin. A, the alignments indicate the amino acid sequences of rat (30), 2A9, rabbit (19), and human platelet calcyclin. B, the alignments indicate the amino acid sequences of bovine aorta calvasculin (21), pEL-98 (22), 18A2 (23), 42A (24), p9Ka (25), and human platelet calvasculin. Asterisks denote amino acid exchanges.

calvasculin, and of proteins whose sequences predicted from their cDNA sequences showed a high homology with bovine calvasculin.

DISCUSSION In this study, using W-7-coupled Sepharose column, we discovered three distinct Ca2+-binding proteins in human platelets. We had already reported that W-7 inhibits platelet aggregation, shape change and secretion associated with interaction with CaM in a Ca2+-dependent manner, and inhibits the CaM-dependent activation of various kinases (13, 14) and myosin phosphorylation. CaM has often been investigated on its

various functions and features, but the other Ca2+-binding proteins have not been studied extensively. Their physiological roles in signal transduction processes remain obscure. 1314

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In the present report, we demonstrated the existence of calcyclin and calvasculin in human platelets. Both belong to the “S-100” protein family. Calcyclin was first identified as the product of 2A9 gene, thereby suggesting that it plays a role in cellcycle regulation (16, 17). At the protein level, the purification

of calcyclin was

reported from Ehrlich ascites tumor cells (18) and from rabbit lung (19). Since calcyclin genes of various species are highly conserved, calcyclin may have an important intracellular function as yet unknown (20). Calvasculin was first identified from the EGTA extract of bovine aorta (21). The partial amino acid sequence of calvasculin revealed a high homology to the proteins encoded by the mRNAs termed pEL-98 (22), 18A2 (23), 42A (24), and p9Ka (25). These mRNAs are abundantly expressed in certain cell lines when they are induced to grow or to differentiate but their function remains unclear. Until now calcyclin and calvasculin have been supposed to be possibly involved in the regulation of the cell proliferation (16, 17, 26, 27). Moreover, the fact that calcyclin and calvasculin exist in anucleate human platelet cytosol can lead to another view of their function. Interestingly, it is reported that calcyclin may regulate the function of a calcyclin-associated protein (CAP-50), a member of the annexin family, in a Ca*+/phospholipid-dependent manner (28), and that placental anticoagulant proteins, members of the annexin family, have potential anticoagulant activities and inhibit phospholipase A2 activity (29). Accordingly, it may be inferred that calcyclin is implicated in hemostasis and that other Ca*+-signaling pathways may well exist in addition to the CaM-mediated signal transduction system. Further investigations of the cellular functions of calcyclin and calvasculin, as well as its regulation by Ca*+, are expected to reveal calcyclin-

and calvasculin-related

intracellular

Ca*+-signal

transduction system in human platelets. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Hathaway,D.R., and Adelstein, R.S. (1979) Proc. Natl. Sci. U.S.A. 76, 1653-1657. Nishikawa, M ., Tanaka,T., and Hidaka, H. (1980) Nature 287, 863-865. Adelstein, R.S., and Eisenberg,E. (1980) Annu. Rev. B&hem. 49,921-956. Nishikawa, M ., Hidaka, H., and Adelstein, R.S. (1983) J. Biol. Chem. 258, 14069-14072. Ferrell, J.E., Martin, G.S. (1988) Mol. Cell Biol. 8, 3603-3610. Siess,G. (1989) Physiol. Rev. 69, 58-178. Levin, R.M., and Weiss, B. (1979) J. Pharmacol. Exp. Ther. 208, 454-459. Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489. 1315

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Watanabe, Y., Usuda, N., Tsugane, S., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem. 267, 17136-17140. Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem. 267, 19596- 19599. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y (1984) Biochemistry 23,50365041. Schlgger, H., and von Jagow, G. (1987) Anal. Biochem. 166,368-379. Hidaka, H., Yamaki, T., Totsuka, T., and Asano, M. (1979) Mol. Pharmacol. 15,49-59. Hidaka, H., Yamaki, T., Naka, M., Tanaka, T., Hayashi, H., and Kobayashi, R. (1980) Mol. Pharmacol. 17, 66-72. Kretsinger, R.H. (1980) CRC Crit. Rev. Biochem. 8, 119-174. Calabretta, B., Kaczmarek, L., Mars, W., Ochoa, D., Gibson, C.W., Hirschhoru, R.R., and Baserga, R. (1985) Proc. Natl. Sci. U.S.A. 82, 4463-4467. Calabretta, B., Battini, R., Kaczmarek, L., deRie1, J.K., and Baserga, R. (1986) J. Biol. Chem. 261, 12628-12632. Kuzunichi, J., and Filipek, A. (1987) Biochem. J. 247,663-667. Tokumitsu, H., Kobayashi, R., and Hidaka, H. (1991) Arch. B&hem. Biophys. 288,202207. Heizmann, C.W., and Hunziker, W. (1990) In Intracellular Calcium Regulation (Brooner, E , ed.), pp. 21 l-248. Wiley Liss,Inc. Watanabe, Y., Kobayashi, R., Ishikawa, T., and Hidaka, H. (1992) Arch. Biochem. Biophys. 292, 563-569. Goto, K., Endo, H.,and Fujiyoshi, T. (1988) J. Biochem. 103, 48-53. Jackson-Grusby, L.L., Swiergiel, J., and Linzer, D.I.H. (1987) Nucleic Acids Res. 15, 6677-6690. Masiakowski, P., and Shooter, E.H. (1988) Proc. Natl. Sci. U.S.A. 85, 1277-1281. Barraclough, R., Savin, J., Dube, S.K., and Rudland, P.S. (1987) J. Mol. Biol. 198, 1320. Ghezzo, F., Lauret, E., Ferrari, S., and Baserga R. (1988) J. Biol. Chem. 263,4758-4763. Ferrari, S., Calabretta, B., deRie1, J.K., Battini, R., Ghezzo, F., Lauret, E., Griffin, C., Emanuel, B.S., Gurrieri, F., and Baserga, R. (1987) J. Biol. Chem. 262, 8325-8332. Tokumitsu, H., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem. 267, 8919-8924. Tait, J.F., Sakata, M., McMullen, B.A., Miao, C.H., Funakoshi, T., Hendrickson, L.E., and Fujikawa, K. (1988) Biochemistry 27,6268-6276. Murphy, L.C., Murphy, L.J., Tsuyuki, D., Duckworth, M.L., and Shiu R.P.C. (1988) J. Biol. Chem. 263, 2397-2401.

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Calcyclin and calvasculin exist in human platelets.

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-7)-coupled Sepharose column, three distinct Ca(2+)-binding proteins have bee...
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