264

BBAPRO 33782

Biochimica et Biophysica Acta, 1041 (1990) 264-268

Elsevier

Boar transition protein 2 and 4 isolated from late spermatid nuclei by high-performance liquid chromatography

Kuniko Akama, Rikako Maruyama, Hikari Mochizuki and Tohru Tobita Department of Chemistry, Faculty of Science, Chiba University, Chiba City (Japan)

(Received 19 March 1990) (Revised manuscript received 23 July 1990)

Key words: Transition protein; High performance liquid chromatography; Late spermatid nuclei; (Boar)

The boar late spermatid nuclei retaining transition proteins (TPs) could be obtained from the testis by the use of antipain to inhibit TP-degrading proteinases of the nuclei. The enzymes detected in acid extract including the basic proteins were inactivated by reduction and carboxymethylation of the proteins. The reduced and carboxymethylated basic proteins were fractionated by differential precipitation between 3% trichioroacetic acid (TCA) and 3-20% TCA. From the 3% TCA-precipitate, boar TP2 and TP4 were isolated by high-pedormance liquid chromatography (HPLC) on Nucleosii 300 7C18. The two TPs were characterized by acid urea- and SDS-polyacrylamide gel electrophoreses and amino acid analysis. Boar TP2 closely resembled rat and mouse TP2s, and ram protein 3 in its high content of serine and basic amino acids, the presence of cysteine, and molecular weight. Boar TP4 was similar to ram protein PI in its high content of basic amino acids, the presence of cysteine and molecular weight. But the TP2 and TP4 differed in electrophoretic mobility on acid urea-gel and solubility in 3% TCA from those of the other species. The HPLC used here also enabled us to efficiently separate boar TPI, TP2, TP3 and TP4, and to estimate that the amount of the TP2, TP3 and TP4 was about 1 / 8 , 1 / 4 and 1 / 4 that of the TP1, respectively.

Introduction

During spermiogenesis in mammals, histones are transiently replaced by several low molecular weight basic proteins called transition proteins, and finally by protamines as the principal basic nuclear proteins of the mature sperm [1-12]. In rats [13] and rams [14], the transition proteins (TPs) are thought to consist of four species. The spermatid nucleus becomes resistant to breakage by sonication shortly after the first TPs appear in the nucleus. Thus sonication is conveniently used for isolation of the nuclei containing TPs [2]. Because the content of basic amino acids of the basic proteins in mammalian spermiogenesis increases in the order his- tones, TPs and mammalian type 1 class of protamines, the electrostatic binding of TPs to DNA would be intermediate between histones and protamines. TP1-4, except for rat and mouse TP1, contain cysteine residues

which are partly cross-linked in chromatin by disulfide bridges [5]. The reconstitution experiments from rat TP1 and nucleic acids suggest that rat TP1 is a DNA- melting protein [15]. Accordingly, the TPs presumably facilitate chromatin transformation from the nucleo- some structure to the nucleoprotamine structure in the replacement of histones by protamines [3,15,16]. Boar TP1 and TP3 have been acid-extracted directly from the testis, and sequenced (Tobita T. et al., unpublished data). However, we did not succeed in preparing boar late spermatid nuclei containing TPs without heat-treat- ment at 100°C for 4 min, because the late spermatid nuclei had TP-degrading activity. In this paper, we describe a method for preparing boar late spermatid nuclei retaining TPs, and for separating boar TP1, TP2, TP3 and TP4, using high-performance liquid chro- matography (HPLC), which enabled us to isolate boar TP2 and TP4 and to estimate the content of these TPs in the late spermatid nuclei.

Abbreviations: TP, transition protein; TCA, trichloroacetic acid; DTT, dithiothreitol.

Correspondence: K. Akama, Department of Chemistry, Faculty of Science, Cltiba University, Chiba City 260, Japan.

Materials and Methods

Materials Bio-Gel P-2 and Nucleosil 300 7C18 were purchased

from Bio-Rad and Chemco, respectively. Otherwise

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

specified, all operations in the following experiments were performed at 4 ° C.

Preparation of boar late spermatid nuclei Boar testis (80 g) was cut into slices with a knife and

suspended in 50 /~M antipain/0.31 M sucrose/3 mM CaC12/1 mM MgC12/10 mM sodium borate buffer (pH 9.0) (240 ml). The suspension was stirred for 30 min and filtered through two layers of gauze, a stainless steel mesh (63 /~m) and a nylon mesh (20 /~m). The filtrate was centrifuged at 2000 × g for 15 rain. The precipitate was suspended in 100 ml 10 mM potassium phosphate buffer (pH 7.4). The suspension was soni- cated on ice twice with 20°s bursts every 60 s at 60-W output, and seven times with 20-s bursts every 60 s at 25-W output, using a Tomy ultrasonic vibrator UR- 200P, thereby disrupting most cellular components ex- cept the late spermatid nuclei. The crude sonication-re- sistant nuclei were recovered by centrifugation at 1500 × g for 30 min, and homogenized in 1.5 M sucrose/1 mM MgC12/10 mM potassium phosphate buffer (pH 7.4) (100 ml) with a teflon homogenizer. The late spermatid nuclei were recovered by centrifugation at 1500 × g for 30 min. The purity of the nuclei prepara- tions was determined by counts made under a phase contrast microscope.

Isolation of transition proteins from boar late spermatid nuclei

Basic proteins were extracted from the nuclei with 0.2 M H2SO 4 (30 ml). The proteins were precipitated from the extract by addition of 80% TCA to give a final concentration of 20%. The precipitate was washed with acidified acetone. The basic proteins were reextracted from the precipitate with 0.9 M acetic acid (30 ml), and the extract was lyophilized. The lyophilized proteins were dissolved in 2.0 ml 6 M guanidine-HC1/20 mM EDTA/0.5 M Tris-HC1 (pH 8.5), containing a 100-fold molar excess of dithiothreitol (DTT) over half-cystine of the proteins under a nitrogen barrier. After the solution had been incubated for 2 h at 37 ° C, a 2.4-fold molar excess of sodium iodoacetate with respect to the DTT was added. After a 2 h-incubation at 37 ° C, the proteins were desalted through Bio-Gel P-2 in 0.2 M acetic acid. The fraction passed through the column was lyophi- lized. The lyophilized proteins were dissolved in 1.5 ml 0.2 M acetic acid. An equal vol. of 6% TCA was added to the solution, and the suspension was allowed to stand at 0 ° C for 30 min and centrifuged at 15 000 × g for 10 rnin. To the supernatant 80% TCA was added to bring the TCA-concentration to 20%, and the precipitate was recovered by centrifugation. These two precipitates were separately washed with acidified acetone and dried. The dried proteins were dissolved in 0.68 M 2-mercapto- ethanol/7 M urea/0.9 M acetic acid, incubated at 37 °C for 1 h, and subjected to reverse-phase HPLC on

265

Nucleosil 300 7C18. The proteins in each peak were lyophilized and redissolved in 0.1% trifluoroacetic acid. Aliquots were used for amino acid and gel electro- phoretic analyses.

Amino acid analysis Samples were hydrolysed in vacuo with 6 M HC1 at

l l 0 ° C for 24 h. Amino acids were determined on a Hitachi 655 amino acid analyser.

Gel electrophoresis Acid-urea polyacrylamide gel electrophoresis (AU-

PAGE) was performed on 15% gel by the method of Panyim and Chalkley [17]. SDS-polyacrylamide gel elec- trophoresis (SDS-PAGE) was performed at pH 8.8 on 15% gel by the method of O'Farrel [18] using calf thymus histone HI (M r 22000), RCM-boar TP3 (M r 9192) and RCM-boar TP1 (M r 6277) as protein markers. The gel was stained with 0.05% Coomasie brilliant blue R-250, destained as described by Fairbanks et al. [19], and scanned with a Shimadzu Dual-wavelength TLC scanner CS-910.

Results and Discussion

Isolation of boar TP2 and 4 Boar TP1 and TP3 were extracted directly with acid

from the testis or the spermatid nuclei that had been boiled for 4 rain essentially by the same method as described by Kistler [20]. These findings suggest that boar late spermatid nuclei have TP-degrading pro- teinases, which may be due to contamination from the acrosome. The activity was found to be inhibited with antipain (50 /tM). Thus we obtained the boar sonica- tion-resistant late spermatid nuclei retaining the TPs using antipain. Because antipain, a noncovalent inhibi- tor, was dissociated from the proteinases at acidic pH, the TP-degrading activity was again detected in the basic proteins extracted with acid from the late sperma- tid nuclei. It was inhibited by reduction and carboxy- methylation of the proteins. The reduced and carboxy- methylated (RCM) proteins were fractionated by dif- ferential precipitation with 3% TCA and 3-20% TCA. The precipitated proteins were separated by HPLC on Nucleosil 300 7C18. Fig. 1 shows a typical elution pattern of the 3% TCA-precipitate. Aliquots of the combined fractions under each peak were subjected to gel electrophoretic and amino acid analyses. The AU- PAGE of the proteins in peaks l a - l d are shown in the bottom of Fig. 1. Table I shows amino acid composi- tions of the proteins in peaks l a - ld . The 3-20% TCA- precipitate were analysed by the same procedure as with the 3% TCA-precipitate. Those results are shown in Fig. 2 and Table II.

The already-known boar TP1 (Ref. 21 and Tobita, T. et al., unpublished data) was eluted in peak la (Fig. 1)

266

and 2a (Fig. 2), and the known boar TP3 (Tobita T. et al., unpublished data) in peak lc (Fig. 1) and 2b (Fig. 2). They were verified by A U - P A G E of the proteins in the respective peaks (Fig. 1B and 2B). The still-ad- sorbed histones were co-eluted with 80% acetonitrile (data not shown).

The peak lb-protein containing cysteine was rich in serine and basic amino acids (Table I). It migrated as a single band, near the core histones in A U - P A G E (Fig. 1B) and between the histone H1 and H3 in SDS-PAGE (Fig. 3A). Its molecular weight was estimated to be 14 600. From these respects, the peak lb-protein closely resembles rat and mouse TP2s [16,22], and ram protein

I

o

Ib Ic

I 2 0 30 TIME (mln)

J

L. 40

}0

7 ,o;

(A2)

S

$

2aL (Ib) TP2

l lie)

lid3 TP4

1

TP2 - - TP4

--TP3

1 2 3 4 5 6 7 8

--TP1

(+)

1

(-)

Fig. 1. (A1) Separation of the 3% TCA-precipitated basic proteins from boar late spermatids by reverse-phase HPLC. The basic protein (1.9 nag) was chromatographed on a column of (4 × 150 rnm) Nucleo- sil 300 7C18 in 0.1% trifluoroacetic acid with a 0-80% acetonitrile (MeCN) linear gradient at 35 ° C. Fractions (1 ml) were collected at a flow rate of 1 ml/min. (B) A acid-urea (6.4 M) gel ¢lectrophoreto- gram of the proteins in peaks of (A1). Lanes: 1, calf thymus whole histones; 2, 3% TCA-precipitate; 3, peak la-proteins; 4, peak lb-prc~ tein; 5, peak lc-proteins; 6, peak ld-protein; 7, boar RCM-TP3; and 8, boar RCM-TP1. (A2) Microdensitometric tracings of lanes

3-6 of (B).

TABLE I

Amino acid composition of proteins in peaks from HPLC of 3% TCA- precipitated basic proteins on nucleosil 300 7C18

The amount of each amino acid is expressed as tool/100 mol and is the average value calculated from duplicate analyses. CM, carboxy- methyl.

Residues la lb lc ld

CM-Cys 3.4 5.2 0.7 1.8 Asp 5.5 5.8 3.2 6.5 Thr 2.9 4.5 7.0 7.0 Ser 17.0 18.8 9.0 6.5 Glu 7.5 8.8 7.9 11.7 Pro 0.1 7.3 13.4 7.3 Gly 9.7 6.0 4.7 6.0 Ala 4.6 3.9 5.0 5.2 Val 1.4 2.4 4.3 4.3 Met 0.5 1.0 0.5 1.5 lie 0.5 1.3 2.5 2.4 Leu 3.2 2.5 2.4 2.5 Tyr 2.7 2.0 2.4 1.7 Phe 3.0 0.0 1.9 0.9 Lys 14.1 8.1 16.6 17.5 His 5.3 6.8 2.0 0.9 Arg 18.8 18.0 16.6 16.1

3 (corresponding to ram TP2) [14]. The cysteine-con- taining peak 1b-protein is not a degradation product of the histone H3 because of its lower electrophoretic mobility than that of the histone H3 in SDS-PAGE (Fig. 3A). Accordingly, we referred to the peak lb-pro- tein as boar TP2.

The peak ld-protein also migrated as a single band in A U - P A G E and SDS-PAGE (Fig. 1B and 3A). Its molecular weight was estimated to be 16000 (Fig. 3B). It is similar to ram protein P1 (corresponding to ram TP4) in high content of basic amino acids, the presence of cysteine (Table I) and molecular weight. It is also not a degradation product of the histone H3 by the same reason as for the peak lb-protein. Thus, we referred to the peak ld-prote in as boar TP4.

According to their designation, boar TPs were eluted separately in the order TP1, TP2, TP3 and TP4, faster than the histones from the column of Nucleosil 300 7C18. Hence this HPLC system using Nucleosil 300 7C18 is a useful method for separating boar TP1, TP2, TP3 and TP4.

Content of boar T P 1 - 4 in the late spermatid nuclei In the HPLC of the 3% TCA-precipitated proteins

(Fig. 1), the TP2 and TP4 were separated as a single protein whose amount was determined by amino acid analysis. By contrast, the TP1 and TP3 were not com- pletely separated f rom the other proteins. The amount of each TP was estimated from the ratio of the TP to the total proteins in the peak: the total proteins were quantified by amino acid analysis (Tables I and II). The ratio was calculated from the peak areas of the TP and

i 0 to)

i i i)

2a

.t 0 Io

8O

,i" o 4%

o

I ~J

. . . . . 2b- - -

20 3(3 40 TIME (rain)

(A2) , ~ 2a)

:2b)

TP3

(+) (B)

H1

H 3 ~ H2A

H2E~ ~

H4 ~

i i I I

i i

-- TP3

--TPI

1 2 3 4 5 6 7

Fig. 2. (A1) Separation of the 3-20% TCA-precipitated basic proteins from boar late spermatid nuclei. The proteins (0.42 mg) was chro- matographed under the same conditions as in Fig. 1 (A1). (B) Acid- urea (6.4 M) gel electrophoretogram of the proteins in peaks of Fig. 2(A1). Lanes; 1, calf thymus whole histones; 2, RCM-whole basic proteins from the late spermatid nuclei; 3, 3-20% TCA-precipitate; 4, peak 2a-proteins; 5, peak 2b-proteins; 6, boar RCM-TP3; and 7, boar

RCM-TP1. (A2) Microdensitometric tracings of lanes 4-5 of (B).

A B

1 2 3 4

(-)

~o - H I ~ 2 0

H3 i ~/H2B - H 2A I 0 - H 4 (+) 5

0 0.5 1.0 Rf

Fig. 3. SDS-polyacrylamide gel electrophoresis of the protein in peak lb and ld. (A) Lanes; 1, boar RCM-TP1 and TP3; 2, peak lb-pro- tein; 3, peak ld-protein; and 4, calf thymus whole histones. (B) Semi-logarithmic plot of molecular weights versus electrophoretic mobilities. RF, electrophoretic mobilities relative to the tracking dye,

Bromophenol blue.

267

TABLE II

Amino acid composition of proteins in peaks from HPLC of 3 -20% TCA-precipitated basic proteins on nucleosil 300 7C18

The amount of each amino acid is expressed as mol/100 mol and is the average value calculated from duplicate analyses.

Residues 2a 2b

CM-Cys 1.6 1.7 Asp 6.7 6.2 Thr 3.2 6.0 Ser 10.1 8.3 Glu 4.6 9.6 Pro 15.3 14.5 Gly 8.5 8.4 Ala 6.3 6.5 Val 2.5 3.6 Met 0.9 1.5 lie 0.0 2.7 Leu 3.1 4.7 Tyr 1.4 2.2 Phe 0.0 2.3 Lys 18.0 13.9 His 3.8 1.5 Arg 14.2 6.5

other proteins in the microdensitometric tracings of the gel (Figs. 1 (A2) and 2 (A2)): the ratio of the TP1 in peak la (Fig. 1) and peak 2a (Fig. 2) were 0.13 and 0.73, respectively; the ratio of the TP3 in peak lc (Fig. 1) and peak 2b (Fig. 2) were 0.71 and 0.52, respectively. The insoluble basic proteins remaining after the three acid extractions were reduced and carboxymethylated, and then re-extracted with acid, containing negligible amounts of TP1-4 (data not shown). Hence TPs were almost completely recovered by the first three acid extractions. The recoveries of boar TP1, TP2, TP3 and TP4 in the chromatographic procedure were 67, 75, 78 and 75%, respectively.

As shown in Table III, the TP1 was the major TP in boar late spermatid nuclei as well as in rat and ram late spermatid nuclei. The amounts of boar TP2, TP3 and TP4 were about 1/8, 1 /4 and 1/4 that of boar TP1, respectively. These ratios may reflect the mechanism of

TABLE III

Content of transition proteins in 3 % TCA- and 3 - 20 % TCA-precipi- tates of basic proteins from boar late spermatid nuclei

The values are normalized to 100 g of the testis and corrected for chromatographic recoveries. See the text on the method for calculat- ing the amount of each TP. ppt, precipitate.

TP1 TP3 TP2 TP4

3% TCA ppt (nmol) 0,9 4.2 4.3 8.3 3-20% TCA ppt (nmol) 33.7 2.9 Total (nmol) 34.6 7.1 4.3 8.3 10-18 mol/nucleus 5,8 1.2 0.7 1.4

268

boar

i

H3-~= H4-

r a m ra t

i i I

= -=

I

I

..... ,TPI. t=m,TP2. ~ , ' I ' P3 . ".~,TP4.

Fig. 4. Schematic electrophoretic mobilities of boar, ram and rat TPs relative to histones H3 and H4 in 15% acid urea (2.5 M) gel.

the chromat in t ransformat ion f rom nucleosome struc- ture to nucleoprotamine structure.

The yield of the boar sonication-resistant late spermatid nuclei (puri ty 88%) was 6 .0 .107 nuclei per g of the testis. This value is the same order as that of rat late spermatid nuclei. Considering the recovery per- centage of 67, the yield of TP1 in boars and rats f rom 10 g of the testis was almost the same [4].

Comparison of the TPs between the species Table I I I shows the results of the differential precipi-

tat ion with 3% T C A and 3-20% T C A of boar TPs. Since boar TP1 in the 3-20% TCA-precipi ta te accounts for 98% of the total TP1, boar TP1, like rat and ram T P l s [3-5], was essentially soluble in 3% TCA; boar TP2 was insoluble in 3% TCA, but rat and ram TP2s are soluble [3,5]; rat and ram TP3s are soluble in 3% T C A [3,5], whereas boar TP3 was distr ibuted almost evenly between the 3% TCA-precipi ta te and the 3-20% TCA-precipitate. This distr ibution is due to its solubil- i ty under the experimental condit ions used here; boar TP4 was insoluble in 3% T C A as well as rat TP4 [23], but ram TP4 is soluble [5].

Mobilities of the boar T P 1 - 4 relative to histones H3 and H4 in A U - P A G E remained unchanged at the urea concentrat ions of 2.5 and 6.4 M (data not shown). Fig. 4 shows the schematic electrophoretic mobilities of boar, ram and rat TPs relative to histones H3 and H4 in acid-urea (2.5 M) PAGE. The boar, ram and rat T P l s migrate similarly [3,5,9,14]. Al though TP2 and TP4 in boars, rams and rats migrate close to the core histones, their mobilities vary slightly with the species [3,14]. Boar and ram TP3s migrate similarly, and faster than rat TP3 [3,14].

Almost the same electrophoretic mobil i ty and the TCA-solubi l i ty of the T P l s are based on that the amino acid sequences of boar, rat, mouse and h u m a n T P l s are highly conserved [20,21,24-26]. On the other hand, mouse TP2 is homologous to rat TP2 on the basis of similarities in amino acid composit ion, molecular weight and the sequence of 27 residues f rom the carboxyl

terminus [16,20]. However, the TP2s as well as the TP3s and TP4s are dissimilar between the species in mobili ty in A U - P A G E and solubility in 3% TCA, suggesting that their amino acid sequences vary from species to species. Fur ther s tudy on the pr imary structure of the TPs is necessary to approve of this presumption.

A c k n o w l e d g e m e n t s

The authors are indebted to Professor Kohsaku M a r u y a m a and Dr. Sumiko Kimura of Chiba Univer- sity for sonication treatment. The authors are also inde- bted to Dr. Shigeru Ohmori of Chiba University for densi tometric analyses.

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