Vol.
182,
No.
February
3, 1992
14,
BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
Pages
1992
PURIFICATION AND CHARACTERIZATION
1246-1253
OF A NEW MEMBER OF THE S-l 00
PROTEIN FAMILY FROM HUMAN PLACENTA
Yutaka Emoto$, Ryoji Kobayashi, Hajime Akatsuka and Hiroyoshi Hidaka Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan SDepartment of Internal Medicine, Branch Hospital of Nagoya University School of Medicine, Higashi-ku, Nagoya 461, Japan Received
December
26,
1991
Summary: A novel Ca*+-binding protein which is termed S-1OOP was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-1OOP. Isoelectric point of S-1OOP was pI=4.6. S-1OOP did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-1OOP was determined. It is 49% identical with human S-lOOp, 38% with human calcyclin, and 37% with human cystic fibrosis antigen. 0 1992 Academic Press,
Inc.
Intracellular Caz+ is known to be a second messenger of various cellular actions such as contraction, secretion, cell growth, differentiation, and neural excitability. Ca2+binding proteins mediate these events. These proteins have homologous sequences, the so-called EF-hand structures (1). Twenty-two EF-hand subfamilies have been identified (2). Calmodulin, troponin C, calbindins, parvalbumin, and S-100 proteins are typical of these subfamilies. Calmodulin is known to regulate numbers of enzymatic activities. Calmodulin exists in all four kingdoms; fungi, protista, plants, and animals, whereas the S-100 protein family and some other low molecular weight Ca2+-binding proteins are expressed in specific tissues in animals. This suggests that the S-100 protein family has specific functions associated with certain differentiated cells. S-100 protein, the first member of the family, was initially found in brain (3). More than 10 years later, two distinct subunits were resolved (4). The other members were The abbreviations used are: kDa, kilodalton( SDS, sodium dodecyl sulfate; CAPS, 3-cyclohexylaminopropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance ’ . chromatography; [ethylenebis(oxyethylenenitrilo)]tetraac~~~~id; 2-ME, 2-mercaptoethano?l$TTFAA. Trifluoroacetic acid; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvimlideni difluoride. 0006-291W92 Copyright All rights
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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ICaBP (5); p10 (calpactin I light chain) (6,7); 2A9 (calcyclin) (8); cystic fibrosis antigen (9); MRP-14 (10); p9Ka (1 l), alternatively termed pEL98 (12), 18A2 (13), or 42A (14); SlOOL (15); and calgizzarin (16). All of these proteins have EF-hand structures. Some of the biological properties of the S-100 protein family were reported. For example, S-1OOp extends neurites in vitro suggesting that S-100 proteins may have a role of messengers between cells (17). S-100 protein inhibits microtuble assembly in a Ca2+ dependent manner (18). PlO and S-1OOp inhibit the phosphorylation of p36 (calpactin I) (6). But the physiological roles of the S-100 protein family in intact cells are not yet elucidated. In surveying S-100 protein family in human placenta, we detected two low molecular weight Ca2+-binding proteins, calcyclin and a new member of the family, S-1OOP. They were homologous to human S-loop. Ca2+ binding abilities and Ca2+-dependent conformational changes of the proteins were examined along with other biochemical characteristics. Materials
and Methods
Materials -All chemicals were of reagent grade. S-100 proteins were prepared from bovine brain by the method of Endo et al (19) followed by reverse phase HPLC (20). Polyacrylamide gel electrophoresis -Proteins were separated by polyacrylamide (10%) gel electrophoresis as described by Schggger and von Jagow (21) in the presence or absence of 2-mercaptoethanol (2-ME). Gels were stained with Coomassie Brilliant Blue R-250. Low Range SDS-PAGE Molecular Weight Standards (BIO-RAD) were used for molecular mass determinations. Immunoblotting -Polyclonal antibodies were raised in rabbits by injecting 1 mg of purified human calcyclin with complete Freund’s adjuvant at first and 1 mg calcyclin with incomplete Frcund’s adjuvant after four weeks. Serum was collected one week after the second injection. The anti-human caicyclin polyclonal antibodies were affinity purified by the method of antibody exchange immunochemistry as described by Hammarback and Vallee (22). Proteins were separated by Tricine-SDS PAGE and to polyvinilidene difluoride (PVDF) membrane (Millipore.) using a BIO-RAD Transblot SD semidry transfer cell. A transfer buffer contained 10 mM 3cyclohexylaminopropanesulfonic acid (CAPS) (pH 11), 10 96 methanol (23). The antibodies were detected by horseradish peroxidase-linked anti-rabbit IgG purchased from Medical & Biological Labs (Nagoya Japan). Peroxidase activity was detected by the oxidative coupling reaction of N,N’-dimethyl-p-phenylendiamine and 4-chloro-lnaphthol as described by Kobayashi and Tashima (24). Analytical geljiltration HPLC -Stokes radii of the proteins measured by analytical gel filtration HPLC (Shimadzu). TSK G3000SW (Tosoh) column was used. Solvent contained 50 mM Tris-HCl (pH 7.5). 100 mM NaCl, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). The total flow rate was 1.0 ml/min. The chromatogram was monitored at 280 nm absorption. Gel filtration calibration kit (Pharmacia); bovine serum albumin as 35.5 A, ovalbumin as 30.5 A and chymotrypsinogen A as 16.4 A, was used as the standard. 4%22+ overluy autoradiography -Calmodulin, calcyclin, S-1OOP from human placenta, and S-100 from bovine brain were run on Tricine-SDS PAGE and to PVDF membrane. 45Ca autoradiography was done by the method of Maruyama et al. (25). The buffer contained 10 mM immidazole-HCl (pH 6.8), 65 mM KCl. Radioactivities were detected by Fuji BAS2000 Bio-imaging analyzer. Ureukzlkalinegel electrophoresis-Urea/alkaline gel electrophomsis was performed as described by Head and Perry (26). Gels were stained with Coomassie Brilliant Blue R250. UV difference absorbance spectrometry -The buffer contained 20 mM histidineKOH (pH 6.8), 0.1 M KCl, 0.2 mM EGTA. After baseline calibration, final concentration 5 mM CaC12 was added to the sample cell and the same amount of distilled
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water was added to the reference cell. Then UV difference absorbance spectra were recorded on a Shimadzu UV-2200 spectrophotometer over the wave length range 245 300 nm with 1 cm path-length cells. Isoelectric focusing -Isoelectric point was measured with AMPHOLINE PAGPLATE, pH 3.5 - 9.5 from LKB. IEF STANDARDS from BIO-RAD was used for isoelectric point determination. Gels were stained with Coomassie Brilliant Blue R-250. Amino acid sequence analysis -Two purified 10 kDa proteins were digested with lysyl endopeptidase (Wake Chemicals) and V8 protease (Takara Biomedicals). Buffer conditions were 50 mM Tris-HCl (pH 7.5), 2 M urea for lysyl endopeptidase, 100 mM ammonium bicarbonate for V8 protease. After incubation at 37 “C for four hours (lysyl endopeptidase) or for twenty hours (V8 protease), Trifluoroacetic acid (TFA) was added to each peptide solution at final concentration of 0.1 %. Peptides were separated by reverse phase HPLC (ODS-M column: Shimadzu). The solvent system was solvent A (0.1 % TFA) and solvent B (0.09% TFA, 80% acetonitrile). A linear gradient from 0% to 100% B in 70 min was used. Total flow rate was 1.O ml/min. Absorbance was monitored at 215 nm. The proteins and the selected peptides were analyzed by Edman degradation and phenylthiohydantoin-amino acids analysis on an automated protein sequencer (model 473A: Applied Biosystems).
Results
and Discussion
Five hundred grams of full-term human placenta was homogenized in 1500 ml of a buffer consisting of 20 mM Tris-HCl (pH 7.5), 0.2 mM of EGTA, 0.1 mM phenylmethylsulfonyl
fluoride
(PMSF) in a Waring
blender. The homogenate was
centrifuged at 10000 g for 40 min. CaC12 and NaCl were added to the supematant at final concentrations of 2.0 mM and 500 mM, respectively. The solution was centrifuged again at 10000 g for 40 min. The supematant in the presence of Ca2+ was applied to a phenylSepharose
CL-4B
preequilibrated
(Pharmacia)
column
with 20 mM Tris-HCl
(50x100
mm) at 4 ‘C,
that had been
(pH 7.5), 500 mM NaCl, 2.0 mM CaC12. The
column was washed extensively with the preequilibrating buffer. Presumptive calciumbinding proteins were eluted with the buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5 mM EGTA. Fractions of 10 ml were collected. Fractions from number 13 to 21 were pooled and dialyzed against 20 mM Tris-HCl (pH 7.5), 1 mM EGTA, 5 mM 2-ME. The protein solution was applied to Momo-Q (Pharmacia) column (1 ml) on FPLC system (Pharmacia LKB Biotechnology) in the presence of 20 mM Tris-HCl (pH 7.5), 1 mM EGTA, 5 mM 2-ME. Proteins were eluted with a linear gradient from 0 M to 0.3 M of NaCl. Total flow rate was 1.0 ml/min. Fractions of 1 ml were collected and analyzed by Tricine-SDS PAGE. We obtained two 10 kDa proteins. By partial amino acid sequencing the 10 kDa protein in fraction number 17 was revealed as calcyclin, a product of a growth-regulated gene. The 10 kDa protein in fraction number 23 was a newly identified protein termed S-1OOP. The 30 kDa protein in the fraction number 44 was thought to be a chorionic gonadotropin, judging from the partial sequence of the protein (data not shown). The 20 kDa protein in fraction number 47 was calmodulin (Fig. 1 a, b). Calcyclin bound to the anti-human calcyclin antibody. The 20 kDa protein in the same lane was a disulfide-linked dimer of calcyclin, because the protein co-migrated with monomer calcyclin in the presence of a reducing agent by SDS-PAGE. But S-1OOP did not bind to the antibody (Fig. 1 c, d). Final purification step of the S-1OOP was
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0.4
E 4
0.3
ii 8 0.2 %m 9 0.1 0.0
b
MMST A
d
C
13
16
17
23
44
47
MMST 17
23
MMST 17
23
Fie. 1, Separation by anion-exchange chromatography and immunological identification of calcyclin. (a) The eluation profile of Mono-Q column (5x50 mm) on FPLC system. Proteins were eluted with a linear gradient of 0 to 0.3 M NaCl. (b) Tricine-SDS PAGE analysis of selected fractions obtained from the Mono-Q column. Ten ~1 of each fraction was applied. The numbers indicate the fraction number of the chromatography. “MMST” indicates molecular mass standards. “A” indicates applied protein solution. Phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) were used as the molecular mass standards. (c) TricineSDS PAGE of molecular mass standards, fraction number 17, number 23. (d) Immunological identification of calcyclin-like protein. The same proteins and amount as (c) were separated by Tricine-SDS PAGE and transblotted to PVDF membrane. Antihuman calcyclin polyclonal antibodies were used. The antibodies were detected by horseradish peroxidase-linked anti rabbit IgG.
performed by HPLC (Shimadzu) with a reverse phasecolumn (Waters mBondapak Cl 8). The solvent systemwas solvent A (0.1 % TFA) and solvent B (0.09% TFA, 80% acetonitrile). A linear gradient from 0% to 100% B in 70 min was used.The total flow The fractions 17 and 23 with Guanidine HCl and TFA at final concentrations of 4 M and 0.1 %, respectively, were applied to HPLC. The chromatogramswere monitored at 215 nm absorption. The calcyclin-like protein was eluted at 59 % acetonitrile concentration, and the S-lOO-like protein at 61 %. The calcyclin forms a disulfide-linked dimer that is detectableby CoomassieBrilliant Blue rate was 2.0 ml/min.
stain of SDS-PAGE, but the S-1OOPdoesnot. The peak fractions
were collected,
freeze-
dried, and dissolvedwith 100ml of distilled water. Two respectiveproteinswere over 95
% pure by Tricine-SDS PAGE(Fig. 2 a. b). Molecular massesof the S-1OOPwere determined as 10 kDa by SDS-PAGE. Stokes radius of S-1OOPwas 25.3 A by analytical gel filtration HPLC. If the protein was globular, molecular massof the protein was 33000. This value was larger than predicted when the protein was globular andexisted asa monomerform. Thus, it might be rod-like or exist asoligomersin native condition.The isoelectricpoint of S- 1OOPwaspI=4.6. 1249
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a
20
40
80
0
% Acelonitrile
20
40 70 Acetonitrile
Fig. 2. Final purification step of calcyclin and S-1OOP by reverse phase high performance liquid chromatography. Reverse phase HPLC eution profile of the fraction number17 in Fig.1 is shownin (a) and number23 in (b). The peak fractionswerecollectedmanuallyandfreezedried.InsetsshowTricine-SDSPAGE of eachprotein with (+2ME) or without (-2MJZ)reducingagent.One p1of peakA was appliedto laneA. Three~1of peakB wasappliedto laneB. “St” indicatesmolecular massstandards whicharethesameasFig.1. Calcium binding abilities of the S-1OOPwas proved by 45Ca2+overlay technique. Ca2+bound to the S-lOOP,the calcyclin, S-100, and calmodulin in the buffer containing 10 mM immidazole-HCl (pH 6.8). 65 mM KCl. (Fig. 3). Considering the protein concentration applied on the gel, radioactivity on the calmodulin band was the highest among the four. The lowest radioactivity was detected on the calcyclin band (Fig. 3). This is probably becauseof their different affinities for Ca2+. Ca2+ dependent conformational change was proved by urea/alkaline gel electrophoresis and UV difference spectrometry. Urea/alkaline gel electrophoresis revealed that the mobilities of S-lOOP, calcyclin, and S-100 protein decreasedin the presenceof Caz+, indicating that the conformational changeof the S-1OOPfrom bovine brain is similar to that of S-100 and calcyclin. On the other hand, calmodulin showed increasedmobility in the presenceof Ca2+(data not shown).UV difference spectrometry of the calcyclin showedtwo positive Ca2+ dependentabsorptionchangesat 282 nm and 288 nm. And that of the S-1OOPshowed single negative changeat 295 nm. The C!az+ a
b
12345
1 2 3 4 5 -45Ca autoradiography of S-lOOP, calcyclin, S-100, and calmodulin. (a) Coomassie Brilliant Blue stainof Tricine-SDSPAGE. Lane 1: molecularweight standardswhich are the sameas Fig.1, lane 2: humancalmoclulin,lane 3: human calcyclin. lane4: humanS-lOOP,lane5: bovineS-100.(b) 45Caautoradiography. The lanesandtheamountof eachproteinarethesameasfor(a). 1250
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io % Acetonitrile
&I
v2
I 40
% Acetonitrile
(“121
a
Fig. 4. Peptide maps and amino acid sequenceof S-1OOPin comparison with other membersof S-100 protein family. Purified S-1OOPwas digestedby aminoacid residue-specificendopeptidases. The namesof the peptidesindicatethe digestingenzymes(L: lysyl endopeptidase; V: staphylococcus aureusV8 protease)and the orderof the HPLCretentiontime.(a) Peptidemapof S-1OOP whichwasdigestedby lysyl endopeptidase. (b) Peptidemapof S-1OOP which wasdigestedby staphylococcus aureusV8 protease.(c) Amino acidsequences of S-1OOP andsomeothermembers of S100proteinfamily. A bar underthe peptidenameindicatesthe rangeof a determined sequence of S-1OOP. The sequence “D” indicatesthedirectsequence of theprotein.The sequences of peptidesaregiven in single-lettercodes.Amino acidresiduesidenticalto thoseof S-1OOPare includedin the boxes.The dotted aminoacid residuesare the putativeCa*+bindingsitesof EF-handstructure.
dependentabsorptionchangesaround 280 nm suggested the positional change of benzene ring in the molecules. The difference between the proteins was probably due to the different amount and location of tyrosine residues in the molecule. (data not shown).
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The S-1OOP was analyzed directly by an automated protein sequencer. It could be sequenceddirectly. This suggestedthat the N-terminal of the protein was not blocked. The purified S- 1OOPwas digestedby lysyl endopeptidaseand staphylococcusaureusV8 protease.Peptideswere resolved by reversephaseHPLC (Fig. 4 a, b). Selectedpeptides were analyzed by an automatedprotein sequencer.Fifteen peptidesof the S-1OOPwere sequenced.None of thesepeptideswas identical to reported proteins in NBRF or Swissprot data bases.A ninety amino acid sequenceof the S-1OOPwere determined(Fig. 4 c). Generally, it is difficult to determine N-terminal and C-terminal of a protein by amino acid sequenceanalysis. Becausemost of the N-terminal of proteins were blocked, and could not be sequenceddirectly. Fortunately, S-1OOPcould be sequenceddirectly from the N-terminal. As for the C-terminal, the determined sequenceof S-1OOPendedwith lysine (K in single-letter code). We used lysyl endopeptidaseto obtain the last peptide (Ll) (Fig. 4). This endopeptidasecleaves the carboxyl sideof lysine, so it is not certain whether this is the C-terminal of the protein or not. The total molecular massof the 90 amino acids was calculated at over 10 kDa, which is very close to the molecular mass obtainedby SDS-PAGE. We concludedthat more than 90 % of the entire sequenceof the protein was determined. S-IOOP has two putative Caz+ binding structures in its sequence.The amino acid sequenceof S-1OOPis 49% identical with human S-loop, 38% with human calcyclin, 37% with humancystic fibrosis antigen, and 46% with pig calpactin I light chain. S-1OOPis lessthan 50 % identical with other membersof the S100 protein family (Fig. 4). Consideringthe S-100 protein family are highly conserved between species,S-1OOPis not identical to any other reported membersof the S-100 family. S-1OOPis a new member of S-100 protein family that is expressedin human placenta.
References 1. Kretsinger, R. H. (1980) C. R. C. Crit. Rev. Biochem. 8, 119-174. 2. HeIemann, C.W. (1991) Novel Calcium-Binding Proteins, pp. 17-37, Spring-Verlag 3. Moore, B. (1965) Biochem. Biophys. Res. Comm. 19,793-744. 4. Isobe, T., Nakajima, T., and Okuyama, T. (1977) Biochem. Biophys. Acta. 494,222-232. 5. Szebenyi, D. M., and Obendorf, S. K. (1981) Nature. 294, 327-332. 6. Gerke, V., and Weber, K. (1985) EMBO J. 4,2917-2920. 7. yt8ney, J. R. Jr., and Tack, B. F. (1985) Proc. Natl. Acad. Sci. USA. 82,78848. Calabretta, B., Battini, R., Kaczmarek, L., de Riel, J. K., and Baserga,R. (1986) J. Biol. Chem. 261,12628-12632. 9. Dorln, J. R., Novak, M., Hill, R. E., Brock, D. J. H., Secher, D. S., and van Heyningen, V. (1987) Nature. 326,614-617. 10. Odink, K., Cerletti, N.,Brtlggen, J., Clerc, R. G., Tarcsay, L., Zwadlo, G., Gerhards, G., Schlegel, R., Sorg, C. (1987) Nature. 330,80-82. 11. Barraclough, R., Savin, J.. Dube, S. K., and Rudland, P. S. (1987) J. Mol. Biol.
198,13-20.
12. Goto,K., Endo, H., and Fujiyoshi, T. (1988) J. Biochem. 103,48-53. 13. Jackson-Grubsy, L. L., Swiergiel. J., and Linzer. D.I.H. (1987) Nucleic Acids Res.
15,6677-6690.
14. Masiakowski, P., and Shooter, E.M. (1988) Proc. Natl. Acad. Sci. USA. 85,12771281. 1252
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15. Glenny, J. R. Jr., Kindy, M. S., and Zokas, L. (1989) J. Cell Biol. 108,569-578. 16. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem.in press. 17. Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. USA. 82,71367139. 18. Endo, T., and Hidaka, H. (1983) FEBS Lerter 161,235-238. 19. Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) 1. Biol. Chem. 256,12485-12489. 20. Isobe, T., Ishioka,N., Masuda, T., Takahashi, Y., Gonno, S., and Okuyama, T. (1983) Biochem. Int. 6,419~426. 21. Schggger, H., and von Jagow, G. (1987) Analytical Biochem. 166,368-379. 22. Hammarback, J. A., and Vallee, R. B. (1990) J. Biol. Chem. 265,12763-12766. 23. Matsudaira, P. (1987) J. Biol..Chem. 262,10035-10038. 24. Kobayashi, R., and Tashima, Y. (1989) Anal, Biochem. 183,9-12. 25. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. 95,5 1 l-5 19. 26. Head, J. F., and Perry, S. V. (1974) Biochem. J. 137,145-154.
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Pages
1992
PURIFICATION AND CHARACTERIZATION
1246-1253
OF A NEW MEMBER OF THE S-l 00
PROTEIN FAMILY FROM HUMAN PLACENTA
Yutaka Emoto$, Ryoji Kobayashi, Hajime Akatsuka and Hiroyoshi Hidaka Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan SDepartment of Internal Medicine, Branch Hospital of Nagoya University School of Medicine, Higashi-ku, Nagoya 461, Japan Received
December
26,
1991
Summary: A novel Ca*+-binding protein which is termed S-1OOP was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-1OOP. Isoelectric point of S-1OOP was pI=4.6. S-1OOP did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-1OOP was determined. It is 49% identical with human S-lOOp, 38% with human calcyclin, and 37% with human cystic fibrosis antigen. 0 1992 Academic Press,
Inc.
Intracellular Caz+ is known to be a second messenger of various cellular actions such as contraction, secretion, cell growth, differentiation, and neural excitability. Ca2+binding proteins mediate these events. These proteins have homologous sequences, the so-called EF-hand structures (1). Twenty-two EF-hand subfamilies have been identified (2). Calmodulin, troponin C, calbindins, parvalbumin, and S-100 proteins are typical of these subfamilies. Calmodulin is known to regulate numbers of enzymatic activities. Calmodulin exists in all four kingdoms; fungi, protista, plants, and animals, whereas the S-100 protein family and some other low molecular weight Ca2+-binding proteins are expressed in specific tissues in animals. This suggests that the S-100 protein family has specific functions associated with certain differentiated cells. S-100 protein, the first member of the family, was initially found in brain (3). More than 10 years later, two distinct subunits were resolved (4). The other members were The abbreviations used are: kDa, kilodalton( SDS, sodium dodecyl sulfate; CAPS, 3-cyclohexylaminopropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance ’ . chromatography; [ethylenebis(oxyethylenenitrilo)]tetraac~~~~id; 2-ME, 2-mercaptoethano?l$TTFAA. Trifluoroacetic acid; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvimlideni difluoride. 0006-291W92 Copyright All rights
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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ICaBP (5); p10 (calpactin I light chain) (6,7); 2A9 (calcyclin) (8); cystic fibrosis antigen (9); MRP-14 (10); p9Ka (1 l), alternatively termed pEL98 (12), 18A2 (13), or 42A (14); SlOOL (15); and calgizzarin (16). All of these proteins have EF-hand structures. Some of the biological properties of the S-100 protein family were reported. For example, S-1OOp extends neurites in vitro suggesting that S-100 proteins may have a role of messengers between cells (17). S-100 protein inhibits microtuble assembly in a Ca2+ dependent manner (18). PlO and S-1OOp inhibit the phosphorylation of p36 (calpactin I) (6). But the physiological roles of the S-100 protein family in intact cells are not yet elucidated. In surveying S-100 protein family in human placenta, we detected two low molecular weight Ca2+-binding proteins, calcyclin and a new member of the family, S-1OOP. They were homologous to human S-loop. Ca2+ binding abilities and Ca2+-dependent conformational changes of the proteins were examined along with other biochemical characteristics. Materials
and Methods
Materials -All chemicals were of reagent grade. S-100 proteins were prepared from bovine brain by the method of Endo et al (19) followed by reverse phase HPLC (20). Polyacrylamide gel electrophoresis -Proteins were separated by polyacrylamide (10%) gel electrophoresis as described by Schggger and von Jagow (21) in the presence or absence of 2-mercaptoethanol (2-ME). Gels were stained with Coomassie Brilliant Blue R-250. Low Range SDS-PAGE Molecular Weight Standards (BIO-RAD) were used for molecular mass determinations. Immunoblotting -Polyclonal antibodies were raised in rabbits by injecting 1 mg of purified human calcyclin with complete Freund’s adjuvant at first and 1 mg calcyclin with incomplete Frcund’s adjuvant after four weeks. Serum was collected one week after the second injection. The anti-human caicyclin polyclonal antibodies were affinity purified by the method of antibody exchange immunochemistry as described by Hammarback and Vallee (22). Proteins were separated by Tricine-SDS PAGE and to polyvinilidene difluoride (PVDF) membrane (Millipore.) using a BIO-RAD Transblot SD semidry transfer cell. A transfer buffer contained 10 mM 3cyclohexylaminopropanesulfonic acid (CAPS) (pH 11), 10 96 methanol (23). The antibodies were detected by horseradish peroxidase-linked anti-rabbit IgG purchased from Medical & Biological Labs (Nagoya Japan). Peroxidase activity was detected by the oxidative coupling reaction of N,N’-dimethyl-p-phenylendiamine and 4-chloro-lnaphthol as described by Kobayashi and Tashima (24). Analytical geljiltration HPLC -Stokes radii of the proteins measured by analytical gel filtration HPLC (Shimadzu). TSK G3000SW (Tosoh) column was used. Solvent contained 50 mM Tris-HCl (pH 7.5). 100 mM NaCl, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). The total flow rate was 1.0 ml/min. The chromatogram was monitored at 280 nm absorption. Gel filtration calibration kit (Pharmacia); bovine serum albumin as 35.5 A, ovalbumin as 30.5 A and chymotrypsinogen A as 16.4 A, was used as the standard. 4%22+ overluy autoradiography -Calmodulin, calcyclin, S-1OOP from human placenta, and S-100 from bovine brain were run on Tricine-SDS PAGE and to PVDF membrane. 45Ca autoradiography was done by the method of Maruyama et al. (25). The buffer contained 10 mM immidazole-HCl (pH 6.8), 65 mM KCl. Radioactivities were detected by Fuji BAS2000 Bio-imaging analyzer. Ureukzlkalinegel electrophoresis-Urea/alkaline gel electrophomsis was performed as described by Head and Perry (26). Gels were stained with Coomassie Brilliant Blue R250. UV difference absorbance spectrometry -The buffer contained 20 mM histidineKOH (pH 6.8), 0.1 M KCl, 0.2 mM EGTA. After baseline calibration, final concentration 5 mM CaC12 was added to the sample cell and the same amount of distilled
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water was added to the reference cell. Then UV difference absorbance spectra were recorded on a Shimadzu UV-2200 spectrophotometer over the wave length range 245 300 nm with 1 cm path-length cells. Isoelectric focusing -Isoelectric point was measured with AMPHOLINE PAGPLATE, pH 3.5 - 9.5 from LKB. IEF STANDARDS from BIO-RAD was used for isoelectric point determination. Gels were stained with Coomassie Brilliant Blue R-250. Amino acid sequence analysis -Two purified 10 kDa proteins were digested with lysyl endopeptidase (Wake Chemicals) and V8 protease (Takara Biomedicals). Buffer conditions were 50 mM Tris-HCl (pH 7.5), 2 M urea for lysyl endopeptidase, 100 mM ammonium bicarbonate for V8 protease. After incubation at 37 “C for four hours (lysyl endopeptidase) or for twenty hours (V8 protease), Trifluoroacetic acid (TFA) was added to each peptide solution at final concentration of 0.1 %. Peptides were separated by reverse phase HPLC (ODS-M column: Shimadzu). The solvent system was solvent A (0.1 % TFA) and solvent B (0.09% TFA, 80% acetonitrile). A linear gradient from 0% to 100% B in 70 min was used. Total flow rate was 1.O ml/min. Absorbance was monitored at 215 nm. The proteins and the selected peptides were analyzed by Edman degradation and phenylthiohydantoin-amino acids analysis on an automated protein sequencer (model 473A: Applied Biosystems).
Results
and Discussion
Five hundred grams of full-term human placenta was homogenized in 1500 ml of a buffer consisting of 20 mM Tris-HCl (pH 7.5), 0.2 mM of EGTA, 0.1 mM phenylmethylsulfonyl
fluoride
(PMSF) in a Waring
blender. The homogenate was
centrifuged at 10000 g for 40 min. CaC12 and NaCl were added to the supematant at final concentrations of 2.0 mM and 500 mM, respectively. The solution was centrifuged again at 10000 g for 40 min. The supematant in the presence of Ca2+ was applied to a phenylSepharose
CL-4B
preequilibrated
(Pharmacia)
column
with 20 mM Tris-HCl
(50x100
mm) at 4 ‘C,
that had been
(pH 7.5), 500 mM NaCl, 2.0 mM CaC12. The
column was washed extensively with the preequilibrating buffer. Presumptive calciumbinding proteins were eluted with the buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5 mM EGTA. Fractions of 10 ml were collected. Fractions from number 13 to 21 were pooled and dialyzed against 20 mM Tris-HCl (pH 7.5), 1 mM EGTA, 5 mM 2-ME. The protein solution was applied to Momo-Q (Pharmacia) column (1 ml) on FPLC system (Pharmacia LKB Biotechnology) in the presence of 20 mM Tris-HCl (pH 7.5), 1 mM EGTA, 5 mM 2-ME. Proteins were eluted with a linear gradient from 0 M to 0.3 M of NaCl. Total flow rate was 1.0 ml/min. Fractions of 1 ml were collected and analyzed by Tricine-SDS PAGE. We obtained two 10 kDa proteins. By partial amino acid sequencing the 10 kDa protein in fraction number 17 was revealed as calcyclin, a product of a growth-regulated gene. The 10 kDa protein in fraction number 23 was a newly identified protein termed S-1OOP. The 30 kDa protein in the fraction number 44 was thought to be a chorionic gonadotropin, judging from the partial sequence of the protein (data not shown). The 20 kDa protein in fraction number 47 was calmodulin (Fig. 1 a, b). Calcyclin bound to the anti-human calcyclin antibody. The 20 kDa protein in the same lane was a disulfide-linked dimer of calcyclin, because the protein co-migrated with monomer calcyclin in the presence of a reducing agent by SDS-PAGE. But S-1OOP did not bind to the antibody (Fig. 1 c, d). Final purification step of the S-1OOP was
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0.4
E 4
0.3
ii 8 0.2 %m 9 0.1 0.0
b
MMST A
d
C
13
16
17
23
44
47
MMST 17
23
MMST 17
23
Fie. 1, Separation by anion-exchange chromatography and immunological identification of calcyclin. (a) The eluation profile of Mono-Q column (5x50 mm) on FPLC system. Proteins were eluted with a linear gradient of 0 to 0.3 M NaCl. (b) Tricine-SDS PAGE analysis of selected fractions obtained from the Mono-Q column. Ten ~1 of each fraction was applied. The numbers indicate the fraction number of the chromatography. “MMST” indicates molecular mass standards. “A” indicates applied protein solution. Phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) were used as the molecular mass standards. (c) TricineSDS PAGE of molecular mass standards, fraction number 17, number 23. (d) Immunological identification of calcyclin-like protein. The same proteins and amount as (c) were separated by Tricine-SDS PAGE and transblotted to PVDF membrane. Antihuman calcyclin polyclonal antibodies were used. The antibodies were detected by horseradish peroxidase-linked anti rabbit IgG.
performed by HPLC (Shimadzu) with a reverse phasecolumn (Waters mBondapak Cl 8). The solvent systemwas solvent A (0.1 % TFA) and solvent B (0.09% TFA, 80% acetonitrile). A linear gradient from 0% to 100% B in 70 min was used.The total flow The fractions 17 and 23 with Guanidine HCl and TFA at final concentrations of 4 M and 0.1 %, respectively, were applied to HPLC. The chromatogramswere monitored at 215 nm absorption. The calcyclin-like protein was eluted at 59 % acetonitrile concentration, and the S-lOO-like protein at 61 %. The calcyclin forms a disulfide-linked dimer that is detectableby CoomassieBrilliant Blue rate was 2.0 ml/min.
stain of SDS-PAGE, but the S-1OOPdoesnot. The peak fractions
were collected,
freeze-
dried, and dissolvedwith 100ml of distilled water. Two respectiveproteinswere over 95
% pure by Tricine-SDS PAGE(Fig. 2 a. b). Molecular massesof the S-1OOPwere determined as 10 kDa by SDS-PAGE. Stokes radius of S-1OOPwas 25.3 A by analytical gel filtration HPLC. If the protein was globular, molecular massof the protein was 33000. This value was larger than predicted when the protein was globular andexisted asa monomerform. Thus, it might be rod-like or exist asoligomersin native condition.The isoelectricpoint of S- 1OOPwaspI=4.6. 1249
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a
20
40
80
0
% Acelonitrile
20
40 70 Acetonitrile
Fig. 2. Final purification step of calcyclin and S-1OOP by reverse phase high performance liquid chromatography. Reverse phase HPLC eution profile of the fraction number17 in Fig.1 is shownin (a) and number23 in (b). The peak fractionswerecollectedmanuallyandfreezedried.InsetsshowTricine-SDSPAGE of eachprotein with (+2ME) or without (-2MJZ)reducingagent.One p1of peakA was appliedto laneA. Three~1of peakB wasappliedto laneB. “St” indicatesmolecular massstandards whicharethesameasFig.1. Calcium binding abilities of the S-1OOPwas proved by 45Ca2+overlay technique. Ca2+bound to the S-lOOP,the calcyclin, S-100, and calmodulin in the buffer containing 10 mM immidazole-HCl (pH 6.8). 65 mM KCl. (Fig. 3). Considering the protein concentration applied on the gel, radioactivity on the calmodulin band was the highest among the four. The lowest radioactivity was detected on the calcyclin band (Fig. 3). This is probably becauseof their different affinities for Ca2+. Ca2+ dependent conformational change was proved by urea/alkaline gel electrophoresis and UV difference spectrometry. Urea/alkaline gel electrophoresis revealed that the mobilities of S-lOOP, calcyclin, and S-100 protein decreasedin the presenceof Caz+, indicating that the conformational changeof the S-1OOPfrom bovine brain is similar to that of S-100 and calcyclin. On the other hand, calmodulin showed increasedmobility in the presenceof Ca2+(data not shown).UV difference spectrometry of the calcyclin showedtwo positive Ca2+ dependentabsorptionchangesat 282 nm and 288 nm. And that of the S-1OOPshowed single negative changeat 295 nm. The C!az+ a
b
12345
1 2 3 4 5 -45Ca autoradiography of S-lOOP, calcyclin, S-100, and calmodulin. (a) Coomassie Brilliant Blue stainof Tricine-SDSPAGE. Lane 1: molecularweight standardswhich are the sameas Fig.1, lane 2: humancalmoclulin,lane 3: human calcyclin. lane4: humanS-lOOP,lane5: bovineS-100.(b) 45Caautoradiography. The lanesandtheamountof eachproteinarethesameasfor(a). 1250
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io % Acetonitrile
&I
v2
I 40
% Acetonitrile
(“121
a
Fig. 4. Peptide maps and amino acid sequenceof S-1OOPin comparison with other membersof S-100 protein family. Purified S-1OOPwas digestedby aminoacid residue-specificendopeptidases. The namesof the peptidesindicatethe digestingenzymes(L: lysyl endopeptidase; V: staphylococcus aureusV8 protease)and the orderof the HPLCretentiontime.(a) Peptidemapof S-1OOP whichwasdigestedby lysyl endopeptidase. (b) Peptidemapof S-1OOP which wasdigestedby staphylococcus aureusV8 protease.(c) Amino acidsequences of S-1OOP andsomeothermembers of S100proteinfamily. A bar underthe peptidenameindicatesthe rangeof a determined sequence of S-1OOP. The sequence “D” indicatesthedirectsequence of theprotein.The sequences of peptidesaregiven in single-lettercodes.Amino acidresiduesidenticalto thoseof S-1OOPare includedin the boxes.The dotted aminoacid residuesare the putativeCa*+bindingsitesof EF-handstructure.
dependentabsorptionchangesaround 280 nm suggested the positional change of benzene ring in the molecules. The difference between the proteins was probably due to the different amount and location of tyrosine residues in the molecule. (data not shown).
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The S-1OOP was analyzed directly by an automated protein sequencer. It could be sequenceddirectly. This suggestedthat the N-terminal of the protein was not blocked. The purified S- 1OOPwas digestedby lysyl endopeptidaseand staphylococcusaureusV8 protease.Peptideswere resolved by reversephaseHPLC (Fig. 4 a, b). Selectedpeptides were analyzed by an automatedprotein sequencer.Fifteen peptidesof the S-1OOPwere sequenced.None of thesepeptideswas identical to reported proteins in NBRF or Swissprot data bases.A ninety amino acid sequenceof the S-1OOPwere determined(Fig. 4 c). Generally, it is difficult to determine N-terminal and C-terminal of a protein by amino acid sequenceanalysis. Becausemost of the N-terminal of proteins were blocked, and could not be sequenceddirectly. Fortunately, S-1OOPcould be sequenceddirectly from the N-terminal. As for the C-terminal, the determined sequenceof S-1OOPendedwith lysine (K in single-letter code). We used lysyl endopeptidaseto obtain the last peptide (Ll) (Fig. 4). This endopeptidasecleaves the carboxyl sideof lysine, so it is not certain whether this is the C-terminal of the protein or not. The total molecular massof the 90 amino acids was calculated at over 10 kDa, which is very close to the molecular mass obtainedby SDS-PAGE. We concludedthat more than 90 % of the entire sequenceof the protein was determined. S-IOOP has two putative Caz+ binding structures in its sequence.The amino acid sequenceof S-1OOPis 49% identical with human S-loop, 38% with human calcyclin, 37% with humancystic fibrosis antigen, and 46% with pig calpactin I light chain. S-1OOPis lessthan 50 % identical with other membersof the S100 protein family (Fig. 4). Consideringthe S-100 protein family are highly conserved between species,S-1OOPis not identical to any other reported membersof the S-100 family. S-1OOPis a new member of S-100 protein family that is expressedin human placenta.
References 1. Kretsinger, R. H. (1980) C. R. C. Crit. Rev. Biochem. 8, 119-174. 2. HeIemann, C.W. (1991) Novel Calcium-Binding Proteins, pp. 17-37, Spring-Verlag 3. Moore, B. (1965) Biochem. Biophys. Res. Comm. 19,793-744. 4. Isobe, T., Nakajima, T., and Okuyama, T. (1977) Biochem. Biophys. Acta. 494,222-232. 5. Szebenyi, D. M., and Obendorf, S. K. (1981) Nature. 294, 327-332. 6. Gerke, V., and Weber, K. (1985) EMBO J. 4,2917-2920. 7. yt8ney, J. R. Jr., and Tack, B. F. (1985) Proc. Natl. Acad. Sci. USA. 82,78848. Calabretta, B., Battini, R., Kaczmarek, L., de Riel, J. K., and Baserga,R. (1986) J. Biol. Chem. 261,12628-12632. 9. Dorln, J. R., Novak, M., Hill, R. E., Brock, D. J. H., Secher, D. S., and van Heyningen, V. (1987) Nature. 326,614-617. 10. Odink, K., Cerletti, N.,Brtlggen, J., Clerc, R. G., Tarcsay, L., Zwadlo, G., Gerhards, G., Schlegel, R., Sorg, C. (1987) Nature. 330,80-82. 11. Barraclough, R., Savin, J.. Dube, S. K., and Rudland, P. S. (1987) J. Mol. Biol.
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15. Glenny, J. R. Jr., Kindy, M. S., and Zokas, L. (1989) J. Cell Biol. 108,569-578. 16. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem.in press. 17. Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. USA. 82,71367139. 18. Endo, T., and Hidaka, H. (1983) FEBS Lerter 161,235-238. 19. Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) 1. Biol. Chem. 256,12485-12489. 20. Isobe, T., Ishioka,N., Masuda, T., Takahashi, Y., Gonno, S., and Okuyama, T. (1983) Biochem. Int. 6,419~426. 21. Schggger, H., and von Jagow, G. (1987) Analytical Biochem. 166,368-379. 22. Hammarback, J. A., and Vallee, R. B. (1990) J. Biol. Chem. 265,12763-12766. 23. Matsudaira, P. (1987) J. Biol..Chem. 262,10035-10038. 24. Kobayashi, R., and Tashima, Y. (1989) Anal, Biochem. 183,9-12. 25. Maruyama, K., Mikawa, T., and Ebashi, S. (1984) J. Biochem. 95,5 1 l-5 19. 26. Head, J. F., and Perry, S. V. (1974) Biochem. J. 137,145-154.
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