Sperm-activating peptide type-V (SAP-V), a fifth member of the sperm-activating peptide family, purified from the egg-conditioned media of the heart urchin Brissus agassizii

Comp. Biochem. Physiol. Vol. 102B,No. 4, pp. 691-700, 1992 Printed in Great Britain

0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd

SPERM-ACTIVATING PEPTIDE TYPE-V (SAP-V), A FIFTH MEMBER OF THE SPERM-ACTIVATING PEPTIDE FAMILY, PURIFIED FROM THE EGG-CONDITIONED MEDIA OF THE HEART URCHIN BRISSUS AGASSIZII KEN-ICHI YOSHINO,* TOSHIFUMITAKAO,§ YASUTSUGUSHIMONISHI§and NORIO SUZUKI* II *Noto Marine Laboratory, Kanazawa University, Ogi, Uchiura, Ishikawa, 927-05, Japan. Tel.: 81 (768) 74 1151; Fax: 81 (768) 74 1644; and §Institute for Protein Research, Osaka University, Suita, Osaka, 565, Japan. Tel: 81 (6) 877 5111: Fax: 81 (6) 876 2533 (Received 3 January 1992) Abstract--1. A novel type of sperm-activating peptide named sperm-activating peptide type-V (SAP-V) was isolated from the egg-conditioned media (egg jelly) of the heart urchin Brissus agassizii and the primary structure of the peptide was determined by fast atom bombardment mass spectrometry as follows: Gly~S;ys-Glu~ly-Leu-Phe-His-Gly-Met-431y-Asn-Cys. L

-

J

2. SAP-V and [Met(O)9]SAP-V stimulated the respiration ofB. agassizii spermatozoa with half-maximal concentrations of 0.5 and 0.3 nM, respectively. However, half-maximal stimulation of the sperm respiration required 40 nM of S-carboxymethylated SAP-V. 3. SAP-V induced significant increases in the cyclic AMP and cyclic GMP levels in B. agassizii spermatozoa in a concentration-dependent manner. 4. The addition of SAP-V to B. agassizii spermatozoa resulted in a mobility shift of a major sperm protein (mol. wt from 133,000 to 129,000) on sodium dodecyl sulfate-polyacrylamide gels.

INTRODUCTION Sea urchin egg-conditioned media (egg jelly) contains sperm-activating peptides (SAPs) that activate spermatozoa. In the early 1980s Suzuki et al. (1981) and Garbers et aL (1982) first purified an SAP from the egg jelly of sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus and determined the primary structure to be G l y - P h e - A s p - L e u - A s n G l y - G l y - G l y - V a l - G l y . The peptide was named sperm-activating peptide type-I (SAP-I). Since then, a number of SAPs have been isolated from the egg jelly of 16 species of sea urchins distributed over four taxonomic orders. SAP-I and 36 SAP-I derivatives were isolated from the egg jelly of 10 species of sea urchins belonging to the order Echinoida (Nomura et al., 1983; Shimomura et al., 1986; Suzuki et al., 1988a; Yoshino et al., 1989, 1991b, c). SAP-IIA ( C y s - V a l - T h r - G l y - A l a - P r o - G l y - C y s Val--Gly-Gly-Gly-Arg-Leu-NH2) was obtained from Arbacia punctulata (Suzuki et al., 1984; Yoshino et al., 1991a), and SAP-IIB (Lys-LeuCys-Pro-Gly-Gly-Asn--Cys-Val) and six SAP-lIB derivatives were purified from the egg jelly of Glyptocidaris crenularis and Stomopneustes variolaris (Suzuki et al., 1988b; Yoshino et al., 1991a). A. punctulata, G. crenularis and S. variolaris belong to the order Arbacioida. From the egg jelly of two species of sand dollars (Clypeaster japonicus and Astriclypeus manni) in the order Clypeasteroida, IITo whom correspondence should be addressed. 691

SAP-Ill ( A s p - S e r - A s p - S e r - A l a - G l n - A s n - L e u - I l e Gly) and nine SAP-III derivatives were isolated and sequenced (Suzuki et al., 1987a; Takao et al., 1990; Yoshino et al., 1990b). Recently, SAP-IV ( G l y - C y s - P r o - T r p - G l y - G l y - A l a - V a l - C y s ) was isolated from the egg jelly of Diaderna setosum belonging to the order Diadematoida (Yoshino et al., 1990a). SAPs cause a number of biochemical and physiological events in sea urchin spermatozoa, including stimulation of the respiration and motility, inductions of increases in cellular cyclic A M P (cAMP), cyclic G M P (cGMP) and Ca 2+ levels (Hansbrough and Garbers, 1981a; Schackmann and Chock, 1986), and in intracellular pH through activation of an Na+/H + exchange system (Hansbrough and Garbers, 1981b; Repaske and Garbers, 1983; Lee and Garbers, 1986), and a transient activation of the membrane form of guanylate cyclase and subsequent inactivation of the enzyme with reduction in apparent mol. wt and dephosphorylation (Ward and Vacquier, 1983; Suzuki et al., 1984; Ward et al., 1985b, 1986; Ramarao and Garbers, 1985; Bentley et al., 1986). SAP-I promotes an acrosome reaction in H. pulcherrimus spermatozoa as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate (Yamaguchi et al., 1988; Shimizu et al., 1990). SAP-IIA acts as a potent chemoattractant for spermatozoa o f A. punctulata (Ward et al., 1985a). We have attempted to correlate SAPs that are gene products (Ramarao et aL, 1990) and play important roles in sea urchin fertilization with the taxonomic position of sea urchin species. We have

692

KEN-ICHI YOSHINOel al.

p r o p o s e d a hypothesis that the structure and activity o f SAPs are specific at the ordinal level, i.e. SAPs obtained from the egg jelly o f sea urchin species belonging to one taxonomic order interact with sperm a t o z o a across the species within the same order, but not with s p e r m a t o z o a in the other orders (Suzuki et al., 1982, 1988c; Suzuki, 1989). In this paper, we describe the structure and biological nature o f a novel type o f SAP purified from the egg jelly o f the heart urchin Brissus agassizii belonging to the order Spatangoida. MATERIALS AND METHODS

Experimental animals and chemicals B. agassizff heart urchins, H. pulcherrimus sea urchins and C. japonicus sand dollars were collected along the coast of Toyama Bay near Noto Marine Laboratory. G. crenularis sea urchins were collected along the coast of Mutsu Bay near the Asamushi Marine Biological Station, Tohoku University. D. setosum sea urchins were collected along the Okinawan coast of the East China Sea near the Sesoko Marine Science Center, University of the Ryukyus. Spermatozoa or eggs were obtained by intracoelomic injection of 0.5 M KCI. Spermatozoa were collected as "dry sperm" at room temperature and stored on ice until use. Eggs were collected in filtered sea-water. Aminopeptidase M (APase M) was obtained from Pierce Chemical Co. (Rockford, IL). Staphylococcus aureus V8 protease (protease V8) was purchased from Miles Inc. (Kankakee, IL). Sodium dodecyl sulfate (SDS) (95% pure material), tris(hydroxymethyl)aminomethane (Tris), N-(2acetamido)-2-aminoethanesulfonicacid (ACES), monensin, oligomycin and molecular weight standards for SDS gel electrophoresis (MW-SDS-200 kit) were obtained from Sigma Chemical Co. (St Louis, MO). Acetonitrile (ACN) of high-performance liquid chromatography (HPLC) grade was purchased from Wako Pure Chemical Industries (Osaka, Japan). Ethylenediaminetetraacetic acid (EDTA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), dithiothreitol (DTT), dithioerythritol (DTE) and other reagents of analytical grade were products of Nacalai Tesque, Inc. (Kyoto, Japan). The composition of artificial sea-water (ASW) was 454 mM NaC1, 9.7 mM KCI, 24.9 mM MgC12, 9.6mM CaC12, 27.1 mM MgSO4 and 4.4mM NaHCO3, buffered with 10 mM Tris (pH 8.2) or 10 mM ACES (pH 6.6). SAP-I was synthesized by the Peptide Institute, Inc. (Osaka, Japan). Synthetic SAP-IIA was a generous gift from Dr H. Shimomura. SAP-IIB was synthesized at our laboratory by the liquid-phase method (Suzuki et al., 1988b). SAP-Ill and SAP-IV were purified from the egg jelly of C. japonicus and D. setosum, respectively (Yoshino et al., 1990a, b). Determination o f sperm respiration rates Respiration rates of spermatozoa (47 mg wet wt) were determined polarographically in 3.0 ml of ASW (pH 6.6 and 8.2) at 20°C with or without various agents using a Yanaco PO-100A oxygraph (Yanagimoto Mfg Co., Kyoto, Japan) equipped with a rotating platinum electrode. Determination o f cyclic nucleotide concentrations To determine cAMP and cGMP concentrations, spermatozoa (47 mg wet wt) were incubated in 1.5 ml of ASW (pH 6.6 and 8.2) for 5 sec at 20°C with various concentrations of SAP obtained from B. agassizii egg jelly. The incubation was stopped by addition of 0.5 ml of 40% (w/v) TCA. Samples were then centrifuged at 700g for 20 min. The resulting supernatant fluid was extracted three times with 4.0 ml of ethyl ether to remove TCA. The aqueous layer was lyophilized and the resulting residue was kept at - 2 0 ° C until use. Cyclic nucleotide

concentration was determined by radioimmunoassay with Yamasa cAMP and cGMP assay kits (Yamasa Shoyu, K. K., Chiba, Japan).

Purification o f sperm-activating peptides from the egg jelly o f B. agassizii The egg suspension obtained from 30 females of B. agassizii was adjusted to pH 5.0 with 0.01 N HC1 to solubilize the jelly coat and then centrifuged at 200g for 10 min. The supernatant fluid (2000 ml) was mixed with 2 vol 99% ethanol and then centrifuged at 10,000g for 30 min at 4°C. The resulting supernatant fluid was then concentrated at 50°C in vaeuo, delipidated by chloroform extraction, and lyophilized. The residue was dissolved in 200 ml of deionized and distilled water (DDW) and the solution was filtered with a Millex-GV filter (0.22 #m, Millipore Co., Bedford, MA), and then applied to a reverse-phase (RP) column. Sperm-activating peptides were purified by sequential RP-HPLC with a Shimadzu Model LC-6A chromatography system, using the following programs. Program I. A column (Shim-pack PREP C-8, 5/tm, 20 x 250ram)equilibrated with 5%ACN in 0.1%TFA in DDW was eluted with the equilibration solvent for 15rain and then eluted with 60% ACN in 0.1% TFA for the next 15 rain, at a flow rate of 9.9 ml/min. Program II. A column (Unisil Q C-8, 5#m, 4.6×250mm) equilibrated with 10%ACN in 0.1% TFA was eluted for 10 min with the equilibration solvent, followed by a linear gradient of ACN from 10 to 50% in 0.1% TFA over a 40-min time period, at a flow rate of 1.0 ml/min. Program III. A column (Unisil Q C-8, 5~tm, 4.6 x 250 mm) equilibrated with 5% ACN in 5 mM sodium phosphate (pH 5.7) was eluted for 20 rain with the equilibration solvent, followed by a linear gradient of ACN from 5 to 30% in 5 mM sodium phosphate (pH 5.7) over a 40-min time period, at a flow rate of 1.0 ml/min. Program IV. A column (Unisil Q C-8, 5#m, 4.6 x 250 mm) equilibrated with 5% ACN in 10 mM ammonium acetate (pH 6.8) was eluted with a linear gradient of ACN from 5 to 30% in 10 mM ammonium acetate (pH 6.8) over a 50-min time period, at a flow rate of 1.0 ml/min. The absorbance at 225 nm of the effluent was monitored in a Shimadzu SPD-6V spectrophotometer. Respirationstimulating activity of samples obtained at each purification step on B. agassizii spermatozoa was assayed routinely as described above.

S-Carboxymethylation S-Carboxymethylation of peptide was carried out by the method of Crestfield et al. (1963). Peptide (16 nmol) was dissolved in 83/~1 of a solution containing 5 mM EDTA and 0.5 M Tris (pH 8.6). The solution was flushed by N 2 stream and then incubated for 10 min at room temperature with or without 1% (v/v) 2-mercaptoethanol. Then, 5 mg of iodoacetic acid in 0.1 N NaOH (100#1) was added, and incubated for 10 min at room temperature. S-Carboxymethylated peptide was purified by RP-HPLC using the following program: a column (Unisil Q C-8, 5#m, 4.6 × 250 ram) equilibrated with 10% ACN in 0.1% TFA was eluted with a linear gradient of ACN from 10 to 30% in 0.1% TFA over a 40-min time period, at a flow rate of 1.0 ml/min. Fast atom bombardment (FAB) mass spectrometry FAB mass spectra were obtained in a JEOL JMS-HX100 double-focusing mass spectrometer equipped with a FAB ion source and a collision cell, and were processed in a JEOL JMA-DA5000 data-acquisition system, as described by Takao et al. (1984). FAB mass measurement of reduced peptide was carried out according to the procedure reported by Despeyroux et al. (1991). The amino acid

693

Sperm-activating peptide type-V (SAP-V)

A

B

E

BA-1

~

c to o4 o4

O t- o 10 20 30 40

C

BA-1

I

"BA-2

y 1~0

BA-2

2'0

3'0

E

40

5'0

6'0

0 10 20 30 40 50 60 70

BA-1 f

F BA-2

f

=> rr"

0 10 20 30 40 50 60 70

10 20 30 40 50 60

10 20 30 40 50 60

Retention Time (min) Fig. 1. Purification of B. agassizii egg jelly peptides by RP-HPLC. (A) Elution profile of crude extract on RP-HPLC using Program I. (B) An active fraction indicated as a bar in (A) was fractionated by RP-HPLC using Program II. (C and D) Two active peak fractions (BA-I and 2) indicated by arrows in (B) were purified further by RP-HPLC using Program III. (E and F) Final RP-HPLC chromatograms of active fractions BA-I and -2 (Program IV). sequence of S-carboxymethylated peptide was determined by a collision-induced dissociation (CID) method in FAB mass spectrometry. The daughter-ion spectrum was obtained by CID of a parent ion of the peptide in a linked-scan, where the electric field (E) and magnetic field (B) were changed, keeping the ratio of B/E constant, as described by Takao et al. (1991). The N-terminal sequence of the peptide was determined by a method combining FAB mass spectrometry and enzymatic digestion (Shimonishi et al., 1981; Takao et al., 1983, 1984, 1985). S-Carboxymethylated peptide (20.7#g, 15.4nmol) was digested with APase M (EC3.4.11.2) (0.4#g) or protease V8 (EC3.4.21.19) (0.5/tg) in 1% (w/v) NH4HCO 3 (7/~1, pH 6.8) for 60 min at 37°C, and 0.5/tl aliquot of the digest was submitted to FAB mass spectrometry.

Other experimental methods Amino acid analysis was performed with a Hitachi L-8500 amino acid analyzer after hydrolysis for 20 hr in constantboiling HC1 (5.7 N) at I10°C in vacuo. Samples for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared as described previously (Suzuki et al., 1987b). B. agassizii spermatozoa (47 mg wet wt) were incubated in 1.5 ml of ASW (pH 8.2) for 30 sec at 20°C with or without SAP (2.2/~M) obtained from B. agassizii. The incubation was stopped by addition of 0.5 ml of 40% (w/v) TCA. The suspension was kept for 30rain at 4°C and then centrifuged at 30,000g for 30rain at 4°C. The resulting pellet was resuspended in 3.0 ml of ice-cold 90% (v/v) acetone and centrifuged (30,000g for 30rain at 4°C). This washing was repeated once. The resulting pellet was resuspended in 3.0 ml of ice-cold acetone (100%) and centrifuged at 30,000g for 30 rain at 4°C. The resulting pellet was lyophilized, and the residue was dissolved in 10% SDS (300 #1) and submitted to SDS-PAGE. SDS-PAGE in 5.5% polyacrylamide slab gel was carried out essentially by the method of Laemmli (1970). The gel was silver-stained by the method of Morrissey (1981). Protein was measured by the method of Lowry modified by Schacterle and Pollack (1973) with bovine serum albumin as a standard.

RESULTS

Purification o f sperm-activating peptides The e t h a n o l extract of B. agassizii-solubilized egg jelly was concentrated, delipidated a n d lyophilized as described in Materials a n d M e t h o d s . The residue, which was active in respiratory stimulation, was dissolved in D D W , a n d subjected to R P - H P L C using P r o g r a m I (Fig. IA). Active materials were eluted with 6 0 % A C N in 0.1% T F A , a n d the fraction containing active materials was saved, lyophilized a n d fractionated further by R P - H P L C using P r o g r a m II. Two active peak fractions were o b t a i n e d (Fig. 1B). These fractions were purified further by R P - H P L C using P r o g r a m III a n d then P r o g r a m IV. Two pure peptides (BA-1 a n d -2) were o b t a i n e d in final a m o u n t s o f 347 a n d 637 nmoi, respectively (Fig. IC-F). The a m i n o acid compositions o f BA-1 a n d -2 are s h o w n in Table 1. Table 1. Amino acid compositions of spermacting peptides obtained from the egg jelly of the heart urchin B. agassizii BA-1 BA-2 Cm-BA-2 CmCys 2.07 (2) 1.24 (1) Asp 1.05 (1) 0.97 (l) Glu 1.03 (l) 0.96 (I) 1.01 (l) Gly 3.56 (4) 3.71 (4) 4.20 (4) l/2Cys 1.46 (2) 1.72 (2) 0.42 (1) Met 0.80 (l) 0.90 (1) Leu 1.00 (1) 1.00 (1) 0.97 (1) 1.03 (1) Phe 0.94(1) 0.91 (1) His 0.84(1) 0.91 (1) 1.08 (1) Total (12) (12) (12) Cm-BA-2: S-carboxymethylated BA-2. The composition is shown as a normalized value with Leu to have one residue, and the number in parentheses refers to the number of residues in the peptide found by sequencing.

694

KEN-ICHIYOSmNOet al.

B. agassizii Spermatozoa l pH 6.6 ASW

~ ' ~

b

SAP-I, SAP-IIA, SAP-lIB, SAP-Ill,

pH 8.2 ASW -~ O O

~

"x~ligomycin

-

min Fig. 2. Effects of various agents on the respiration of

B. agassizii spermatozoa (15.7 mg wet wt/ml ASW). Arrows (a, b, c and d) indicate the points at which SAP-V (1.1 #M), monensin (14/tM), various sperm-activating peptides (1.1 #M each of SAP-I, SAP-IIA, SAP-IIB, SAP-III and SAP-IV), and oligomycin (3.3/~g/ml) were added. As shown in Fig. 2, B. agassizii sperm respiration was low at pH 6.6. The peptides purified from B. agassizii egg jelly stimulated the respiration up to the level obtained at pH 8.2. However, the sperm

A

Primary structure o f S A P - V Figure 3A shows the FAB mass spectrum of SAPV in the range from 1100 to 1400 atomic mass units. The observed mass value (1222.1) of the peptide is nearly identical to the theoretical mass value (1222.4) calculated from the amino acid composition of Asn (1), Glu (1), Gly (4), Met (1), Leu (1), Phe (1) and His (1) with one cystine. To confirm the presence of an intramolecular disulfide linkage in the peptide, SAP-V (1 mM) in a reducing buffer (1% NH4HCO3, pH 6.8) was mixed with an equal vol of a reductive matrix

1222.1

.............. ......................,i..¸.....,,,,,.¸

1100 O t-

respiration was not stimulated by SAPs such as SAP-l, SAP-IIA, SAP-IIB, SAP-III and SAP-IV isolated from the egg jelly of sea urchins in the orders Echinoida, Arbacioida, Clypeasteroida and Diadematoida. The B. agassizii egg jelly peptide-stimulated respiration was blocked by oligomycin, suggesting that the stimulated respiration is coupled with ATP production. The peptides did not stimulate the sperm respiration of H. pulcherrimus in the order Echinoida, G. crenularis in the order Arbacioida, D. setosum in the order Diadematoida, and C. japonicus in the order Clypeasteroida. These results show that peptides obtained from B. agassizii are specific for B. agassizii spermatozoa. Since BA-2 was obtained in the largest amount, the peptide was named spermactivating peptide type-V (SAP-V) according to the naming system proposed by Suzuki (1989).

12'00

1300

1400

1300

1400

1224.2

B

t~

t'-s .O <

N ] . . . .

i., . . . . . . .

1100

hi..,~,.

........................................

Iq,h ,.,,. II,.: ...~tl

1200

i

.

1238.2

C

1100

.d....

............... iLL.......... ........................... 1200

1300

.......... 1400

m/z Fig. 3. FAB mass spectra. (A) SAP-V; (B) reduced SAP-V; (C) Met(O)-containing SAP-V.

Sperm-activating peptide type-V (SAP-V)

A

1340.2

...... 1100

O~ O E

695

B

,

...................... 1200

d................ l]................................ 13'00

--CmCys

"1"

Gly

:,,,,ll.......................... L. 1400

-1

1283.2

"o t-

.(3 <

1340.2 i

.>_.

N n,-

1122.11

I

IL .....

.....hLm+Uh+II'..+'+[~+..,,'B~................. IllJ]u,+.,]~,.t:,,I .................[U., Jldl,..,+Jd,,=t,=diE+.............. thl........................... L......................... It.........

1100

C

1200

13'00 1340.2

+

993.3

. . . . . . L __, . . . . 900

1400

1000

i.L.Ata~i..il

1100

1200

~ ..... 1300

1400

m/z

Fig. 4. FAB mass spectra. (A): CM-SAP-V; (B): APase M digest of CM-SAP-V; (C): protease V8 digest of CM-SAP-V. (DTT/DTE, 5:1 w/w), and then submitted to FAB mass measurement. The resulting reduced peptide gave a signal at m/z = 1224.2, which is nearly identical to the theoretical mass value (1224.5) of the peptide containing two cysteine residues instead of one cystine residue (Fig. 3B). SAP-V (16 nmol) was treated with iodoacetic acid (5 mg) in the presence of 1% (v/v) 2-mercaptoethanol. The resulting S-carboxymethylated SAP-V (CM-SAP-V) was subjected to amino acid analysis (Table 1). In the FAB mass spectrum of CM-SAP-V, a prominent pseudomolecular ion was observed at m/z = 1340.2, which is consistent with the theoretical mass value (1340.5)

of the peptide (Fig. 4A). While SAP-V treated with iodoacetic acid in the absence of 2-mercaptoethanol showed the same amino acid composition and mass value as those of the untreated peptide (data not shown). These results also show that SAP-V contains an intramolecular disulfide linkage. In order to determine the amino acid sequence of SAP-V, a CID method in FAB mass spectrometry was applied to CM-SAP-V. The CID spectrum of the peptide obtained by a B/E-linked scanning method showed the type y", and b. series of sequence ions (Fig. 5). From this result, the N- and C-terminal sequences were determined to be

y'

8 <

~=

! L, L L. _£t,.~:1.....L.L........ 1 200 400 600

I:)7

aT I

800

all

b9 b~0. r bll i

10'00

12'00

m/z

Fig. 5. CID/Linked-scan spectrum of CM-SAP-V (m/z = 1340.2) recorded from 200 to 1343 atomic mass units. The observed daughter-ions were assigned as y", and b, series of sequence ions (see Table 2). In addition to these, some of type an sequence ions were observed at m/z = 1133.3(air), 961.9 (ag). 830.5 (as), 773.4 (aT) and 489.0 (as). The nomenclature used for sequence ions is based on a slight variation of the Roepstorff-Fohlman notation (Roepstorff and Fohlman, 1984; Biemann, 1988).

KEN-ICHIYOSHINOet al.

696

Table 2. The sequence ions observed in the CID spectrum of molecular ion (m/z = 1340.2) of CM-SAP-V Ion

Mass value

Difference

Y"H Y"~0 Y"9 Y"8 Y"7 Y"6

Not observed 1122.4 992.9 935.8 822.5 675.1

217.8 129.5 57.1 113.3 147.4

Gly + CmCys Glu Gly Leu Phe

(218.0) (129.0) (57.0) (113.1) (147.1)

bll bt0 b9 b8 b7 b6 b5 b4 b3 b2

1161.5 1047.0 989.9 858.7 801.5 Not observed 517.0 403.8 346.9 218.1

160.7 114.5 47.1 131.2 57.2

CmCys Asn Gly Met Gly

(161.0) (114.0) (57.0) (131.0) (57.0)

284.5 113.2 56.9 128.8

His + Phe Leu Gly Glu

(284.2) (113.1) (57.0) (129.0)

{%)~

Amino acids

"Difference" denotes mass difference between Y"cn + ~) and y", ions, or between bl, + ~1and b n ions, except values of y"~0 and b 5 ions. With regard to Y"~o and b 5 ions, it denotes mass difference between Y"~2 and Y"t0 ions or between b 7 and b 5 ions. The value in parentheses indicates the theoretical residual mass value of the amino acid.

"~_ ~ 50

._g

I

13 12

11 10 9 8 ~' Log [Peptide] (M)

Fig. 6. Concentration-dependent effect of SAP-V and two SAP-V derivatives on B. agassizii sperm respiration. The potency of the peptides in stimulation of the sperm respiration rate is expressed as the ratio of net increased rate to the rate obtained in ASW at pH 8.2. (IN): SAP-V; (I): [Met(O)9]SAP-V; (A): CM-SAP-V.

theoretical mass values (1221.5-1225.4) calculated from the amino acid composition (Fig. 3C). The H-(Gly, CmCys)-Glu-Gly-Leu-Phe- and Glu-Glydifference (15.9) between the observed mass values Leu-(Phe, His)~Gly-Met-Gly-Asn-CmCys-OH, reof BA-1 and SAP-V corresponded to the mass of an spectively (Table 2). Thus, the partial sequence of oxygen atom, suggesting that methionine in BA-1 is CM-SAP-V was determined to be (Gly, CmCys)oxidized to be methionine sulfoxide [Met(O)]. ThereGlu-Gly-Leu-Phe-His-Gly-Met-Gly-Asn-CmCys. fore, we concluded that BA-1 is an SAP-V derivative, To determine the N-terminal sequence of the peptide, [Met(O)9]SAP-V. CM-SAP-V was digested with APase M or protease V8. Figure 4B shows the FAB mass spectrum of Effects o f S A P - V on the heart urchin spermatozoa a digest of CM-SAP-V with APase M for 60min SAP-V stimulated B. agassizii sperm respiration at 37°C. Two intense signals at m / z = 1283.2 and half-maximally at a concentration of about 0.5 nM 1122.1 were observed together with the signal at (Fig. 6). [Met(O)9]SAP-V was also quite a potent m/z = 1340.2 of the undigested peptide. The mass stimulator of B. agassizii sperm respiration. The differences (57.0 and 161.1) between 1340.2 and peptide stimulated the sperm respiration with a half1283.2 and between 1283.2 and 1122.1 corresponded maximal concentration of 0.3 nM, which is comparto the residual mass values of Gly (57.0) and CmCys able to that of SAP-V (Fig. 6). However, CM-SAP-V (161.0). After digestion of CM-SAP-V with protease ([CmCys2.~Z]SAP-V) was a less potent stimulator of V8 for 60 rain at 37°C, a new signal at m/z = 993.3 B. agassizii sperm respiration. The S-carboxywas observed in the FAB mass spectrum (Fig. 4C). methylated peptide stimulated the respiration with The mass difference (346.9) between 1340.2 and 993.3 half-maximal concentration of 40nM (Fig. 6). indicates that a tripeptide which consists of Gly (1), SAP-V induced the increases in the cGMP and CmCys (1) and Glu (1) was released from CM-SAP-V cAMP levels in B. agassizii spermatozoa within by protease V8 treatment. The C-terminus of the 5 sec at pH 6.6 and 8.2 in a concentration-dependent tripeptide should be glutamic acid since protease manner. The increase in the cGMP level occurred V8 cleaves specifically at the COOH side of gluta- at more than 10 nM of SAP-V (Fig. 7), while that mic acid in 1% (w/v) NH4HCO 3 buffer (pH 6.8) of the cAMP level required more than 100nM of (Houmard and Drapeau, 1972). From these results, the peptide (Fig. 8). When B. agassizii spermaN-terminal sequence of CM-SAP-V was determined tozoa were treated with SAP-V (2.2#M) in ASW to be H-Gly-CmCys-Glu-. (pH 8.2), a newly stained protein with a mol. wt Taken together all of evidences described above, of 129,000 appeared, concomitant with the diswe concluded that the primary structure of SAP-V is as follows: Gly-Cys-Glu~;ly-Leu-Phe-His-Gly-Met-Gly-Asn-Cys.

Methionine sulfoxide-containing SAP- V As shown in Table 1, BA-1 had the same amino acid composition as that of SAP-V (BA-2). However, BA-1 and SAP-V were eluted at different retention times in RP-HPLC under the same condition (Fig. 1D and F). Moreover, the FAB mass spectrum of BA-1 gave a pseudomolecular ion signal at m/z = 1238.2, which is not consistent with the

appearance of a protein having a mol. wt of 133,000 (Fig. 9). DISCUSSION

In the present study, we isolated a sperm-activating peptide from the egg jelly of the heart urchin

Sperm-activating peptide type-V (SAP-V)

697

.,.., 750 t-

~ 5(10 E E

~- 25(1

-5

,I

-Log [SAP-V]

5

(M)

Fig. 7. Concentration-dependent effect of SAP-V on increase in the cGMP level in B. agassizii spermatozoa. ( 0 ) and (O) denote the values obtained in ASW at pH 8.2 and 6.6, respectively. B. agassizii belonging to the order Spatangoida, and characterized the chemical and biological nature of the peptide. The primary structure of the peptide was completely different from those of SAPs isolated from the egg jelly of sea urchins in the orders Echinoida, Arbacioida, Diadematoida and Clypeasteroida. The peptide not only stimulated B. agassizii sperm respiration but also induced increases in the cAMP and cGMP levels in the spermatozoa. Furthermore, addition of the peptide to B. agassizii spermatozoa resulted in the appearance of a new protein with an apparent mol. wt of 129,000, estimated by SDS--PAGE under reducing conditions. In this sense, the peptide can be considered to be functionally the same as other SAPs. However, the peptide did not appear to have any respiration-stimulating effect on spermatozoa of H. pulcherrimus, G. crenularis, D. setosum and C. japonicus. Therefore, we concluded that the peptide is a novel type of sperm-activating peptide, i.e. a fifth member of SAP family, and named sperm-activating peptide type-V (SAP.V) according to the naming system proposed by Suzuki (1989). SAPs such as SAP-I, SAP-IIA, SAP-lIB, SAP-Ill

Fig. 9. The effect of SAP-V on the electrophoretic mobility of a high-molecular-weight protein of B. agassizii spermatozoa. Left lane: untreated spermatozoa; right lane: SAP-V (2.2/tM)-treated spermatozoa. Equal amount of protein (15/Jg) was analyzed by SDS-PAGE using 5.5% polyacrylamide slab gel in the presence of SDS and 2mercaptoethanol.

.~6 35 .,~ E 4

6 ~cr~3 E

(3

"5 1 0 I

1~1 1()

I

I

9 8 7 -Log [SAP-V]

I

I

6 5 (M)

Fig. 8. Concentration-dependent effect of SAP-V on increase in the cAMP level in B. agassizi spermatozoa. (O) and (©) denote the values obtained in ASW at pH 8.2 and 6.6, respectively.

and SAP-IV did not stimulate the respiration rate of B. agassizii spermatozoa. This suggests that B. agassizii spermatozoa, which should have a receptor specific for SAP-V, do not have that for SAP-I, SAP-I/A, SAP-lIB, SAP-III, or SAP-IV. This strongly supports a hypothesis that SAPs isolated from the egg jelly of a given sea urchin species belonging to one taxonomic order interact with spermatozoa of other species within the same order, but not with spermatozoa of the species in other orders (Suzuki et al., 1982, 1988c; Suzuki 1989). In addition to SAP-V, we isolated Met(O)-containing SAP-V ([Met(O)9]SAP-V) from the egg jelly of the same species. Methionine is readily oxidized to methionine sulfoxide even in weak acid. Therefore, Met(O)-containing SAP-V may be produced by chemical oxidation of SAP-V somewhere in the purification steps. However, Met(O)-containing SAPV was also quite a potent stimulator of the sperm

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KEN-ICHIYOSHINOet al.

respiration of B. agassizii. In a previous study (Yoshino et al., 1991c), we isolated a Met(O)-containing SAP, G l y - P h e - G l u - M e t ( O ) - G l y - G l y - T h r - G l y Val-Gly ([Glu3, Met(O) 4, Gly 5, Thr7]SAP-I) from the egg jelly of the sea urchin Heterocentrotus marnmillatus. The peptide was a less potent stimulator of H. pulcherrimus sperm respiration. Studies on the structure-activity relationship of SAP-I demonstrated that the aliphatic amino acid residue at position 4 is essential for the activity (Nomura and Isaka, 1985; Nomura, 1987). Therefore, the lower activity of the Met(O)-containing SAP-I derivative may be explained by less hydrophobicity of Met(O) than Met at position 4 of the peptide. In contrast with the SAP-I derivative, oxidation of the Met residue in SAP-V does not alter the activity of the peptide. This suggests that the hydrophobicity at position 9 of SAP-V is not important for the activity of the peptide. However, S-carboxymethylated SAP-V was less potent than untreated SAP-V in stimulation of the respiration of B. agassizii spermatozoa. This suggests that the intramolecular disulfide linkage in SAP-V is essential for the activity. Disulfide linkage is a covalent bond to stabilize the structure of proteins and peptides, and it plays an important role in maintaining secondary and tertiary structures. As with SAP-V, an intramolecular disulfide linkage is found in SAP-IIA, SAP-lIB, six SAP-lIB derivatives and SAP-IV. These peptides were isolated from the egg jelly of sea urchins belonging to the order Arbacioida or Diadematoida (Yoshino et al., 1990a, 1991a). On the other hand, SAP-I and SAP-Ill which were isolated from the egg jelly of species in the orders Echinoida and Clypeasteroida, respectively, do not contain an intramolecular disulfide linkage. In regular echinoids, species in the orders Arbacioida and Diadematoida are considered to be more primitive than those in the order Echinoida. Therefore, the presence of an intramolecular disulfide linkage in SAPs could be considered a primitive character of echinoids. Accordingly, B. agassizii in the order Spatangoida is more primitive than C. japonicus and A. manni in the order Clypeasteroida since SAP-V from B. agassizii contains an intramolecular disulfide linkage, but SAP-Ill and SAP-Ill derivatives from C. japonicus and A. manni do not contain it. This is consistent with paleontological evidence that species in the order Spatangoida appeared at the beginning of the Cretaceous and those in the order Clypeasteroida appeared at the end of the Cretaceous (a time difference of about 50 million years) (Durham 1966a, b; Fischer, 1966). A comparison of the amino acid sequences of SAP-V and other SAPs, such as SAP-I, SAP-IIA, SAP-lIB and SAP-Ill, shows a low degree of similarity, but SAP-V has some similarities in its primary structure to that of SAP-IV, such as Gly~Cys- of the N-terminus and the presence of an intramolecular disulfide linkage formed by cysteine residues at position 2 and the C-terminus. This implies that the ancestor of species in the order Spatangoida arose from species in the order Diadematoida, or that they arose from a common ancestor. Acknowledgements--The authors wish to thank the staff members of the Radioisotope Center, Kanazawa University

for facilitating the use of equipment for determining cAMP and cGMP concentrations. They also express their appreciation to Mr M. Matada for collecting and culturing sea urchins, sand dollars, and heart urchins. They are grateful to the staff members of the Asamushi Marine Biological Station, Tohoku University and the Sesoko Marine Science Center, University of the Ryukyus for their kind help in collecting sea urchins, D. setosum and G. crenularis, respectively. This work was supported in part by Grants-in-Aid (No. 02404006 and 02044059 to N.S.) from the Ministry of Education, Science and Culture of Japan. REFERENCES

Bentley J. K., Tubb D. J. and Garbers D. L. (1986) Receptor-mediated activation of spermatozoan guanylate cyclase. J. biol. Chem. 261, 14859-14862. Biemann K. (1988) Contributions of mass spectrometry to peptide and protein structure. Biomed. environ. Mass Spectrom. 19, 99-111. Crestfield A. M., Moore S. and Stein W. H. (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. biol. Chem. 238, 622-627. Despeyroux D., Bordas-Nagy J. and Jennings K. R. (1991) Determination of the amino acid sequence of cystine-containing peptides by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 5, 156-159. Durham J. W. (1966a) Classification. In Treatise on Invertebrate Paleontology, Part U, Echinodermata 3 (Edited by Moore R. C.), Vol. 1, pp. U270-295, The Geological Society of America and the University of Kansas Press, Kansas. Durham J. W. (1966b) Clypeasteroids. In Treatise on Invertebrate Paleontology, Part U, Echinodermata 3 (Edited by Moore R. C.), Vol. 2, pp. U450-491, The Geological Society of America and the University of Kansas Press, Kansas. Fischer A. G. (1966) Spatangoids. In Treatise on Invertebrate Paleontology, Part U, Echinodermata 3 (Edited by Moore R. C.), Vol. 2, pp. U543 632, The Geological Society of America and the University Kansas Press, Kansas. Garbers D. L., Watkins H. D., Hansbrough J. R., Smith A. and Misono K. S. (1982) The amino acid sequence and chemical synthesis of speract and speract analogues. J. biol. Chem. 257, 2734-2737. Hansbrough J. R. and Garbers D. L. (1981a) Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa. J. biol. Chem. 256, 1447-1452. Hansbrough J. R. and Garbers D. L. (1981b) Sodium-dependent activation of sea urchin spermatozoa by speract and monensin. J. biol. Chem. 256, 2235 2241. Houmard J. and Drapeau G. R. (1972) Staphylococcal protease: A proteolytic enzyme specific for glutamoyl bonds. Proc. natn. Acad. Sci. U.S.A. 69, 3506-3509. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680-685. Lee H. C. and Garbers D. L. (1986) Modulation of the voltage-sensitiveNa +/H + exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract. J. biol. Chem. 261, 16026 16032. Morrissey J. H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem. 117, 307-310. Nomura K. (1987) Structure-activity relationships of the sperm-activating peptides from sea urchin egg jelly: essential roles of the hydrophobic residues. Biochem. Life Sci. Adv., India 6, 9-14. Nomura K. and Isaka S. (1985) Synthetic study on the structure-activity relationship of sperm-activating pep-

Sperm-activating peptide type-V (SAP-V) tides from the jelly coat of sea urchin eggs. Biochem. biophys. Res. Commun. 126, 974-982. Nomura K., Suzuki N., Ohtake H. and Isaka S. (1983) Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthocidaris crussispina. Biochem. biophys. Res. Commun. 117, 147-153. Ramarao C. S. and Garbers D. L. (1985) Receptor-mediated regulation of guanylate cyclase activity in spermatozoa. J. biol. Chem. 260, 8390-8396. Ramarao C. S., Burks D. J. and Garbers D. L. (1990) A single mRNA encodes multiple copies of the egg peptide speract. Biochemistry 29, 3383-3388. Repaske D. R. and Garbers D. L. (1983) A hydrogen ion flux mediates stimulation of respiratory activity by speract in sea urchin spermatozoa. J. biol. Chem. 258, 6025-6029. Roepstorff P. and Fohlman J. (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Muss Spectrom. I1, 601. Schackmann R. W. and Chock B. P. (1986) Alteration of intracellular [Ca2+ ] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca 2+ is coupled to Na ÷ entry and increased intracellular pH. J. biol. Chem. 261, 8719-8728. Schacterle G. R. and Pollack R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Analyt. Biochem. 51, 654-655. Shimizu T., Kinoh H., Yamaguchi M. and Suzuki N. (1990) Purification and characterization of the egg jelly macromolecules, sialoglycoprotein and fucose sulfate glycoconjugate, of the sea urchin Hemicentrotus pulcherrimus. Devl. Growth Diff. 32, 473-483. Shimomura H., Suzuki N. and Garbers D. L. (1986) Derivatives of speract are associated with the eggs of Lytechninus pictus sea urchins. Peptide 7, 491-495. Shimonishi Y., Hong Y.-M., Takao T., Aimoto S., Matsuda H. and Izumi Y. (1981) A new methods for carboxyl-terminal sequence analysis of a peptide using carboxypeptidases and field-desorption mass spectrometry. Proc. Japan Acad. 57B, 304-308. Suzuki N. (1989) Sperm-activating peptides from sea urchin egg jelly. In Bioorganic Marine Chemistry (Edited by Scheuer P. J.), Vol. 3, pp. 47-70. Springer, Berlin. Suzuki N., Nomura K., Ohtake H. and Isaka S. (1981) Purification and the primary structure of sperm-activating peptides from jelly coat of sea urchin eggs. Biochem. biophys. Res. Commun. 99, 1238-1244. Suzuki N., Hoshi N., Nomura K. and Isaka S. (1982) Respiratory stimulation of sea urchin spermatozoa by egg extracts, egg jelly extracts and egg jelly peptides from various species of sea urchins: taxonomical significance. Comp. Biochem. Physiol. 72A, 489-495. Suzuki N., Shimomura H., Radany E. W., Ramarao C. S., Ward G. E., Bentley J. K. and Garbers D. L. (1984) A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa. J. biol. Chem. 259, 14874-14879. Suzuki N., Kurita M., Yoshino K., Kajiura H., Nomura K. and Yamaguchi M. (1987a) Purification and structure of mosact and its derivatives from the egg jelly of the sea urchin Clypeaster japonicus. Zool. Sci. 4, 649-656. Suzuki N., Kurita M., Yoshino K. and Yamaguchi M. (1987b) Speract binds exclusively to sperm tails and causes an electrophoretic mobility shift in a major sperm tail protein of the sea urchin. Zool. Sci. 4, 641-648. Suzuki N., Kajiura H., Nomura K., Garbers D. L., Yoshino K., Kurita M., Tanaka H. and Yamaguchi M. (1988a) Some more speract derivatives associated with eggs of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina. Comp. Biochem. Physiol. 89B, 687-693.

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Yoshino K., Yamaguchi M., Suzuki N., Takao T., Shimonishi Y. and Nomura K. (1991c) Further structural study of sperm-activating peptides isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Heterocentrotus mammillatus and Clypeaster japonicus, using fast atom bombardment mass spectrometry. In Biology of Echinodermata (Edited by Yanagisawa T., Yasumasu I., Oguro C., Suzuki N. and Motokawa T.), pp. 419-425. A. A. Balkema, Rotterdam, The Netherlands.