THE JOURNAL OF EXPERIMENTAL ZOOLOGY 263210-214 (1992)
Xenopws laevis Sperm Proteins, Previously Identified as Surface Proteins With Egg Coat Binding Capability, Are Indeed Histone H4, Histone H3, and Sperm Specific Protein SP2 GIOVANNI BERNARDINI, ISABELLA DALLE DONNE, SABRINA NORRERI, ARMAND0 NEGRI, AND ALDO MILZANI Dipartimento di Biologia (G.B., I.D.D., S.N., A.M.) and Istituto di Fisiologia Veterinaria e Biochimica and CISME (A.N.), Universita di Milano, 1-20133 Milan, Italy ABSTRACT Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88).We have studied the three more abundant ones of apparent molecular weight of 14,19, and 25 kd in SDS-PAGE.We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2. o 1992 Wiley-Liss, Inc.
Sperm-egg interactions that prelude to the fusion of gamete plasma membranes are known to be mediated by the binding of sperm surface ligands to receptors located in the egg coats (Macek and Shurr, '88). Lindsay and Hedrick ('86, '88) have performed a set of experiments aimed at determining which proteins were responsible for sperm-egg interaction in Xenopus. By means of surface iodination techniques they have selected the proteins exposed to the external environment. Among those, by overlay, they have sorted out four sperm proteins of apparent molecular weight of 14, 19, 25, and 35 kd which were capable of binding to molecules of the egg coats. These proteins, therefore, seemed to possess the characteristics to play a cardinal role in initiating fertilization and, because of that, we thought that they deserved attentive studies. Consequently, we have started a further characterization focusing our attention on the three more abundant ones, i.e., 14, 19, and 25 kd (Bernardini et al., '90). To our surprise, we have learned that these proteins are indeed histone H4, histone H3, and sperm-specific protein SP2.
MATERIAL AND METHODS Xenopus sperm suspension was obtained by mincing the testes in the De Boers-Tris solution (DBT) as previously described (Bernardini et al., '89). DBT composition was (in mM) NaClll9, KC1 2.5, CaCI2 1.8, and TrislHCl 15 (pH 7.5). The suspension was first centrifuged (7g, 10 min, 5°C) to remove large tissue fragments and then (l,OOOg, 15 min, 5°C) to 01992 WILEY-LISS,INC.
pellet the spermatozoa. A pair of testes usually gave 80 to 100 x lo6 spermatozoa. For detergent extraction the pellet was solubilized with DBT containing 1%sodium dodecyl sulphate (SDS)for 1to 1.5h in ice and then centrifuged at 30,000 r.p.m. for 45 min at 18°C with a Beckman type 50 Ti rotor (81,400g at r,,,) t o separate soluble from insoluble material (Lindsay and Hedrick, '88). To have a fraction enriched of 14, 19, and 25 kd, the sperm pellet was first solubilized with DBT containing a non-ionicdetergent such as Triton X-100 or Nonidet P-40 for 1 to 1.5 h in ice and then centrifuged at 30,000 r.p.m. for 45 min at 18°C. The supernatant (non-ionic detergent soluble fraction) was discarded and the pellet (non-ionic detergent insoluble fraction) was gently washed with DBT. The pelleted insoluble fraction could now be tested with different zwitter ionic detergents or with increasing SDS concentration. The tested zwitterionic detergents were CHAPS (31(3-cholamidopropyl) dimethylammoniol- 1-propane sulfonate), SB3- 12, C12-C, C12-B, and C l l - 0 (Rabilloud et al., '90). Alternatively, the insoluble fraction could be solubilized directly in SDS-polyacrylamidegel electrophoresis (PAGE)sample buffer. SDS-PAGE was performed according to Laemmli ('70) and protein concentration was determined from gels according to Edgar ('90). For protein purification, the SDS extract was loaded on a 1.5 mm thick preparative 15% SDSReceived April 19,1991;revision accepted January 17,1992.
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PAGE. The gel was run for about 30 rnin after the exit of the bromophenol blue front to achieve a better separation. After a 5 to 15 min staining and a 15 to 30 min destaining, the bands corresponding to 14,19, and 25 kd (Lindsay and Hedrick, '88)were excised and electroeluted with a Biorad Electroeluter model 422. The recovered proteins were precipitated by addition of 100% (wiv) trichloroacetic acid (TCA) to a final concentration of 20%, centrifuged, resuspended, precipitated, washed thoroughly with diethyl ether or with cold ( - Z O O C ) acetone, and dried under vacuum. For acid extraction of basic proteins the method described by Yokota et al. ('91)was followed. Briefly, the sperm pellets were treated with 0.4 N H2S04 and the insoluble material was precipitated by centrifugation. The supernatant was recovered and proteins were TCA precipitated, washed, dried, and analyzed by acid/urea/Triton X-100 (AUT)-PAGE (Zweidler,'78). Gels containing 12% acrylamide, 2.5 M urea, 6 mM Triton X-100, and 5%acetic acid were first preelectrophoresed in 5%acetic acid and then scavenged with 1 M cysteamine HC1 in 5% acetic acid. Samples were dissolved in a small volume of 50% acetic acid and immediately after in sample buffer (5% mercaptoethanol, 8 M urea, 0.01% pyronin Y) and then electrophoresed in 5% acetic acid at 120 V. Gels were stained in 0.05% Coomassie Brilliant Blue R250 (CBB), 10% acetic acid, 50% methanol, and destained i n 7.5%acetic acid and 22% methanol. For two-dimensional electrophoresis, 1 mm thick AUT-PAGE gels were stained for 0.5 min and destained for 2 min; lanes were cut, equilibrated with SDS-PAGE running buffer (1.5-2 h at room temperature), and loaded on the stacking gel of a 1.5 mm thick, 13.5%slab gel for SDS-PAGE. For sequencing, proteins originated from an SDS extract (Lindsay and Hedrick, '88) were electroeluted as described or transferred on Immobilon PDVF membranes (Millipore Corp., Bedford, MA). Following electrophoresis, gels were equilibrated for 20 min in transfer buffer (20% methanol, 25 mM Tris, 192 mM glycine, and 0.07% SDS), blotted for 90 min at 200 mA with a Biorad Trans-Blot Cell, stained for 7 min (0.1% CBB, 50% methanol), destained for 10 rnin (50% methanol, 10% acetic acid), washed with ultrapure water, and dried. Bands of the proteins of interest were excised and introduced in the sequencer (cf. Matsudaira, '87). Sequencing was performed by automated Edman degradation i n a pulse liquid sequencer model 477/A from Applied Biosystem according to manufacturer instructions.
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Fig. 1. Reduced (lane 1) and unreduced (lane 2) sperm extract were separated on a 12% SDS-polyacrylamide gel. Arrowheads point to 14,19, and 25 kd proteins.
RESULTS Xenopus sperm proteins of apparent molecular weight of 14,19, and 25 kd (Lindsay and Hedrick, '86, '88)can be extracted simply by solubilizingXenopus spermatozoa with a solution containing 1% SDS. A successive centrifugation allows to separate a liquid phase from a gelatinous mass of nucleic acids (Lindsay and Hedrick, '88). Figure 1 shows the protein pattern of reduced and unreduced samples on a 12% SDS-PAGE. The non-ionic as well as the zwitter ionic detergents were ineffective in solubilizing the 14, 19, and 25 kd proteins (Fig. 2).We have tested Nonidet P-40 and Triton X-100 and, among the zwitter ionic, the commercially available CHAPS and four other recently synthesized detergents (see Materials and Methods) with improved solubilizing properties (Rabilloud et al., '90). The insolubility of 14, 19, and 25 kd proteins in non-ionic detergents allows a n easy step of enrichment; in fact, the fraction soluble in non-ionic detergent (Fig. 2, lane T) and that one soluble only in SDS (Fig. 2, lanes SDS) are respectively the 49 and 51%of the total protein content; 14, 19, and 25 kd proteins are, respectively, the 12, 15, and 16% of the protein fraction soluble only in SDS (we have measured a protein content of 2.6 mg/108spermatozoa). To determine the appropriate range of SDS concentration needed to solubilize the proteins, we have tested progressively increasing SDS concentrations on the Triton insoluble fraction; the results indicate that 0.1% SDS is sufficient for solubilizing 14, 19, and 25 kd proteins.
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Fig. 2. 13.5%SDS-PAGE.The sperm pellet was solubilized with Triton X-100 (lane T). The zwitterionic detergents C 12-B (lane Z left) and C 12-C (lane Z right) were tried on the Triton insoluble fraction. The insoluble fraction was then solubilized with 1%SDS (lanes SDS). Arrowheads point to 14,19, and 25 kd proteins.
Fig. 3. The SDS extract (i.e., Triton-insoluble, SDS-soluble fraction) is compared to the acid extract on adjacent lanes of a 13.5%SDS-PAGE. The proteins of interest (i.e., those with apparent molecular weight of 14, 19, and 25 kd on SDS-PAGE) are present in both extracts.
Proteins were blotted on Immobilon for direct sequencing or electroeluted and TCA precipitated. The results obtained are shown compared to H3 sequence (Old et al., '85; Perry et al., '85) in Table 1. Sperm basic proteins can be extracted by H2S04 solubilization, precipitated and dried (Yokota et al., '91). If this acid extracted pool of proteins is run on an SDS-PAGE it forms a pattern quite similar to the Triton-insoluble, SDS-soluble fraction (Fig. 3); the proteins with apparent molecular weight of 14, 19, and 25 kd are present also in the HzSO4 extract. The HzSO4 extract is run on an AUT-PAGE where basic proteins are normally separated as histones and protamines (Fig. 4, lane 1).By comparison with the data present in literature (Yokota et al., '91; Mann et al., '82; Kasinsky et al., '85), one can distinguish H3, H4, SP2, SP3-5, and SP6. Electroeluted 14,19, and 25 kd (comingfrom SDS extract) were compared on an AUT-PAGE with an HzSO4 extract; 14 kd migrates as H4 (Fig. 4, lane 2), 19 kd (as expected after amino acid sequenc-
ing) migrates as H3 (Fig. 4, lane 31, and 25 kd migrates as SP2 (Fig. 4, lane 4). Conversely, the H2S04extract (i.e., the extract obtained by the conventional procedure used for basic protein extraction), after a first separation in AUT-PAGE, was run in second dimension in SDS PAGE (Fig. 5).The second dimension shows that histone H4, histone H3, and sperm specific protein SP2 migrates, in SDS-PAGE, as expected for the 14, 19, and 25 kd proteins.
DISCUSSION
Recently, surface proteins of Xenopus spermatozoa which are able to bind to egg envelope components have been identified (Lindsay and Hedrick, '87, '88). We have initiated their characterization as a first step to understand their function. However, these proteins resulted t o be the sperm specific protein SP2 and the histones H3 and H4. Protein 19 kd has been sequenced from Western Blotting and from electroeluted material; both determinations indicate a high identity with histone H3. Moreover, proteins purified from SDS extracts (Lindsay TABLE I . Amino acid sequences of the 19 kdprotein obtained and Hedrick, '88) by electroelution from SDS-PAGE migrate in AUT-PAGE where histones H4 and H3, from blotted (BL) or from electroeluted (EE) material are compared to the sequence of histone H 3 (H3) aspublished by and SP2 of Xenopus are known to migrate. These Old et al. ('85) and Perry et al. ('85) results were confirmed by two-dimensional electrophoresis, where migration patterns due to charge BL A-X-T-K-Q-T-A-X-X-S-T-GEE A-X-T-K-Q-T-A-X-K-S-T-G-G-K-A-P-X-K-Q-L-A-T-K-A-A- (AUT-PAGE)are compared to migration due to mass H3 A-RT-K-Q-T-A-RK-S-T-G-G-K-A-P-RK-Q-L-A-T-K-A-A- (SDS-PAGE).
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Fig. 5 . Two-dimensional PAGE. The acid extract was run for the first dimension in an AUT-PAGE and, for the second dimension, on a 13.5% SDS-PAGE. Arrowheads point to H3, H4, and SP2 that migrate in the second dimension as expected for the 19,14, and 25 kd proteins; 0,origin.
Fig. 4. Proteins coming from an SDS extract and electroeluted from a preparative SDS-PAGE were compared to a sperm acid extract (lane 1)on an AUT-PAGE. Arrowheads of lane 1 point (from top to bottom) to H3, H4, SP2, SP3-5, and SP6. Arrowheads of lane 2 to 4 point, respectively, to electroeluted 14, 19, and 25 kd proteins. The electrophoresis direction was from top ( + ) to bottom ( - ).
Histones and sperm specific basic proteins are known to be localized into the nucleus of mature Xenopus spermatozoa (Moriya and Katagiri, '91). How, then, to explain the experiments of Lindsay and Hedrick ('88)that show that these proteins were 1)surface proteins and 2) able to bind components of the egg coats? The results of the iodination experiments may have suffered from technical problems such as a too harsh iodination; alternatively, we should consider the exiting possibility of nuclear protein exposed t o the external environment. The ability of these proteins to bind to egg coat components is easier to explain as it could be caused by non-specific bindings due to the highly basic nature of these proteins. The discrepanciesbetween the molecular weights of histones H4 and H3 (i.e., 11and 15 kd) and those determined by SDS-PAGE (i.e., 14 and 19 kd) are explained by the exceptional basicity of these proteins and by the consequently reduced overall negative charge. It is well known, in fact, that histones, when analyzed by SDS-PAGE, migrate slower than expected and consequently their molecular weight will be overestimated if compared to non-basic proteins (Hames, '81). Similarly, we ought to forecast a reduced molecular weight for the 25 kd also.
ACKNOWLEDGMENTS This work was supported by an M.P.I. grant. The authors are pleased to thank Dr. E. Gianazza for the gift of the zwitterionic detergents. LITERATURE CITED Bernardini, G., G. Zanmarchi, I. Dalle Donne, and A. Milzani (1990) Three major components of Xenopus spermatozoon plasma membrane. Cell Biol. Int. Rep., 14:167 (abstract). Bernardini, G., G. Zanmarchi, and P. Belgiojoso (1989) The plasma membrane of Xenopus laevis spermatozoon. Gamete Res.,24:237-246. Edgar, A.J. (1990) Gel electrophoresis of native gelsolin-actin complexes. J. Muscle Res. Cell Motil., 11:323-330. Hames, B.D. (1981) An introduction to polyacrylamide gel electrophoresis. In: Gel Electrophoresis of Proteins. B.D. Hames and D. Rickwood, eds. IRL Press, Oxford. Kasinsky, H.E., S.Y. Huang, M. Mann, J . Roca, and J.A. Subirana (1985) On the diversity of sperm histones in vertebrates. IV. Cytochemical and amino acid analysis in Anuru. J . Exp. Zool., 234:33-46. Laemmli, U.K. (1970)Cleavage of structural proteins during the assemblyofthe head ofbacteriophageT4. Nature, 277:680-685. Lindsay, L.L., and J.L. Hedrick (1986) Identification of spermenvelope binding components in Xenopus laevis gametes by Western blotting. Dev. Growth Diff., 28:102 (abstract). Lindsay, L.L., and J.L. Hedrick (1988) Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes. J. Exp. Zool., 245:286-293. Macek, M.B., and B.D. Shur (1988) Protein-carbohydrate complementarity in mammalian gamete recognition. Gamete Res.,20:93-109. Matsudaira, l? (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem., 262:10035-10038. Mann, M., M.S. Risley, R.A. Eckhardt, and H.E. Kasinsky (1982) Characterization of spermatidhperm basic chromosomal proteins in the genus Xenopus (Anura, Pipidae). J . Exp. Zool., 222: 173- 186.
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Moriya, M., and C. Katagiri (1991) Immunoelectron microscopic localization of sperm specific nuclear basic proteins during spermatogenesis in anuran amphibians. Dev. Growth Diff., 33:19-27. Old, R.W., S.A. Sheikh,A. Chambers, C.A. Newton, A. Mohammed, and T.C. Aldridge (1985) Individual Xenopus histone genes are replication-independent in oocytes and replicationdependent in Xenopus or mouse somatic cells. Nucl. Acids a s . , 13:7341-7358. Perry, M., G.H. Thomsen, and R.G. Roeder (1985) Genomic organization and nucleotide sequence of two distinct histone gene clusters from Xenopus laeuis. J. Mol. Biol., 185:479-499.
Rabilloud, T., E. Gianazza, N. Cattb, and P.G. Righetti (1990) Amidosulfobetaines, a family of detergents with improved solubilization properties: Application for isoelectric focusing under denaturatingconditions. Anal. Biochem., 185:94-102. Yokota, T., K. Takamune, and C. Katagiri (1991) Nuclear basic proteins ofXenopus laeuis sperm: Their characterisation and synthesis during spermatogenesis. Dev. Growth Diff. 33: 9-17. Zweidler, A. (1978) Resolution of histones by polyacrylamide gel electrophoresis in presence of non ionic detergents. In: Methods in Cell Biology. G. Stein, J. Stein, and L.J. Kleinsmith, eds. Academic Press, New York, Vol. 17, pp. 223-233.
Xenopws laevis Sperm Proteins, Previously Identified as Surface Proteins With Egg Coat Binding Capability, Are Indeed Histone H4, Histone H3, and Sperm Specific Protein SP2 GIOVANNI BERNARDINI, ISABELLA DALLE DONNE, SABRINA NORRERI, ARMAND0 NEGRI, AND ALDO MILZANI Dipartimento di Biologia (G.B., I.D.D., S.N., A.M.) and Istituto di Fisiologia Veterinaria e Biochimica and CISME (A.N.), Universita di Milano, 1-20133 Milan, Italy ABSTRACT Recently, four Xenopus sperm proteins thought to be involved in binding to the egg envelope were identified (Lindsay and Hedrick, J. Exp. Zool., 245:286-293, '88).We have studied the three more abundant ones of apparent molecular weight of 14,19, and 25 kd in SDS-PAGE.We have shown that these proteins are indeed nuclear basic proteins: the 14 kd is the histone H4, the 19 kd is the histone H3, and the 25 kd is the sperm-specific protein SP2. o 1992 Wiley-Liss, Inc.
Sperm-egg interactions that prelude to the fusion of gamete plasma membranes are known to be mediated by the binding of sperm surface ligands to receptors located in the egg coats (Macek and Shurr, '88). Lindsay and Hedrick ('86, '88) have performed a set of experiments aimed at determining which proteins were responsible for sperm-egg interaction in Xenopus. By means of surface iodination techniques they have selected the proteins exposed to the external environment. Among those, by overlay, they have sorted out four sperm proteins of apparent molecular weight of 14, 19, 25, and 35 kd which were capable of binding to molecules of the egg coats. These proteins, therefore, seemed to possess the characteristics to play a cardinal role in initiating fertilization and, because of that, we thought that they deserved attentive studies. Consequently, we have started a further characterization focusing our attention on the three more abundant ones, i.e., 14, 19, and 25 kd (Bernardini et al., '90). To our surprise, we have learned that these proteins are indeed histone H4, histone H3, and sperm-specific protein SP2.
MATERIAL AND METHODS Xenopus sperm suspension was obtained by mincing the testes in the De Boers-Tris solution (DBT) as previously described (Bernardini et al., '89). DBT composition was (in mM) NaClll9, KC1 2.5, CaCI2 1.8, and TrislHCl 15 (pH 7.5). The suspension was first centrifuged (7g, 10 min, 5°C) to remove large tissue fragments and then (l,OOOg, 15 min, 5°C) to 01992 WILEY-LISS,INC.
pellet the spermatozoa. A pair of testes usually gave 80 to 100 x lo6 spermatozoa. For detergent extraction the pellet was solubilized with DBT containing 1%sodium dodecyl sulphate (SDS)for 1to 1.5h in ice and then centrifuged at 30,000 r.p.m. for 45 min at 18°C with a Beckman type 50 Ti rotor (81,400g at r,,,) t o separate soluble from insoluble material (Lindsay and Hedrick, '88). To have a fraction enriched of 14, 19, and 25 kd, the sperm pellet was first solubilized with DBT containing a non-ionicdetergent such as Triton X-100 or Nonidet P-40 for 1 to 1.5 h in ice and then centrifuged at 30,000 r.p.m. for 45 min at 18°C. The supernatant (non-ionic detergent soluble fraction) was discarded and the pellet (non-ionic detergent insoluble fraction) was gently washed with DBT. The pelleted insoluble fraction could now be tested with different zwitter ionic detergents or with increasing SDS concentration. The tested zwitterionic detergents were CHAPS (31(3-cholamidopropyl) dimethylammoniol- 1-propane sulfonate), SB3- 12, C12-C, C12-B, and C l l - 0 (Rabilloud et al., '90). Alternatively, the insoluble fraction could be solubilized directly in SDS-polyacrylamidegel electrophoresis (PAGE)sample buffer. SDS-PAGE was performed according to Laemmli ('70) and protein concentration was determined from gels according to Edgar ('90). For protein purification, the SDS extract was loaded on a 1.5 mm thick preparative 15% SDSReceived April 19,1991;revision accepted January 17,1992.
XENOPUS LAEVIS SPERM PROTEINS
PAGE. The gel was run for about 30 rnin after the exit of the bromophenol blue front to achieve a better separation. After a 5 to 15 min staining and a 15 to 30 min destaining, the bands corresponding to 14,19, and 25 kd (Lindsay and Hedrick, '88)were excised and electroeluted with a Biorad Electroeluter model 422. The recovered proteins were precipitated by addition of 100% (wiv) trichloroacetic acid (TCA) to a final concentration of 20%, centrifuged, resuspended, precipitated, washed thoroughly with diethyl ether or with cold ( - Z O O C ) acetone, and dried under vacuum. For acid extraction of basic proteins the method described by Yokota et al. ('91)was followed. Briefly, the sperm pellets were treated with 0.4 N H2S04 and the insoluble material was precipitated by centrifugation. The supernatant was recovered and proteins were TCA precipitated, washed, dried, and analyzed by acid/urea/Triton X-100 (AUT)-PAGE (Zweidler,'78). Gels containing 12% acrylamide, 2.5 M urea, 6 mM Triton X-100, and 5%acetic acid were first preelectrophoresed in 5%acetic acid and then scavenged with 1 M cysteamine HC1 in 5% acetic acid. Samples were dissolved in a small volume of 50% acetic acid and immediately after in sample buffer (5% mercaptoethanol, 8 M urea, 0.01% pyronin Y) and then electrophoresed in 5% acetic acid at 120 V. Gels were stained in 0.05% Coomassie Brilliant Blue R250 (CBB), 10% acetic acid, 50% methanol, and destained i n 7.5%acetic acid and 22% methanol. For two-dimensional electrophoresis, 1 mm thick AUT-PAGE gels were stained for 0.5 min and destained for 2 min; lanes were cut, equilibrated with SDS-PAGE running buffer (1.5-2 h at room temperature), and loaded on the stacking gel of a 1.5 mm thick, 13.5%slab gel for SDS-PAGE. For sequencing, proteins originated from an SDS extract (Lindsay and Hedrick, '88) were electroeluted as described or transferred on Immobilon PDVF membranes (Millipore Corp., Bedford, MA). Following electrophoresis, gels were equilibrated for 20 min in transfer buffer (20% methanol, 25 mM Tris, 192 mM glycine, and 0.07% SDS), blotted for 90 min at 200 mA with a Biorad Trans-Blot Cell, stained for 7 min (0.1% CBB, 50% methanol), destained for 10 rnin (50% methanol, 10% acetic acid), washed with ultrapure water, and dried. Bands of the proteins of interest were excised and introduced in the sequencer (cf. Matsudaira, '87). Sequencing was performed by automated Edman degradation i n a pulse liquid sequencer model 477/A from Applied Biosystem according to manufacturer instructions.
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Fig. 1. Reduced (lane 1) and unreduced (lane 2) sperm extract were separated on a 12% SDS-polyacrylamide gel. Arrowheads point to 14,19, and 25 kd proteins.
RESULTS Xenopus sperm proteins of apparent molecular weight of 14,19, and 25 kd (Lindsay and Hedrick, '86, '88)can be extracted simply by solubilizingXenopus spermatozoa with a solution containing 1% SDS. A successive centrifugation allows to separate a liquid phase from a gelatinous mass of nucleic acids (Lindsay and Hedrick, '88). Figure 1 shows the protein pattern of reduced and unreduced samples on a 12% SDS-PAGE. The non-ionic as well as the zwitter ionic detergents were ineffective in solubilizing the 14, 19, and 25 kd proteins (Fig. 2).We have tested Nonidet P-40 and Triton X-100 and, among the zwitter ionic, the commercially available CHAPS and four other recently synthesized detergents (see Materials and Methods) with improved solubilizing properties (Rabilloud et al., '90). The insolubility of 14, 19, and 25 kd proteins in non-ionic detergents allows a n easy step of enrichment; in fact, the fraction soluble in non-ionic detergent (Fig. 2, lane T) and that one soluble only in SDS (Fig. 2, lanes SDS) are respectively the 49 and 51%of the total protein content; 14, 19, and 25 kd proteins are, respectively, the 12, 15, and 16% of the protein fraction soluble only in SDS (we have measured a protein content of 2.6 mg/108spermatozoa). To determine the appropriate range of SDS concentration needed to solubilize the proteins, we have tested progressively increasing SDS concentrations on the Triton insoluble fraction; the results indicate that 0.1% SDS is sufficient for solubilizing 14, 19, and 25 kd proteins.
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Fig. 2. 13.5%SDS-PAGE.The sperm pellet was solubilized with Triton X-100 (lane T). The zwitterionic detergents C 12-B (lane Z left) and C 12-C (lane Z right) were tried on the Triton insoluble fraction. The insoluble fraction was then solubilized with 1%SDS (lanes SDS). Arrowheads point to 14,19, and 25 kd proteins.
Fig. 3. The SDS extract (i.e., Triton-insoluble, SDS-soluble fraction) is compared to the acid extract on adjacent lanes of a 13.5%SDS-PAGE. The proteins of interest (i.e., those with apparent molecular weight of 14, 19, and 25 kd on SDS-PAGE) are present in both extracts.
Proteins were blotted on Immobilon for direct sequencing or electroeluted and TCA precipitated. The results obtained are shown compared to H3 sequence (Old et al., '85; Perry et al., '85) in Table 1. Sperm basic proteins can be extracted by H2S04 solubilization, precipitated and dried (Yokota et al., '91). If this acid extracted pool of proteins is run on an SDS-PAGE it forms a pattern quite similar to the Triton-insoluble, SDS-soluble fraction (Fig. 3); the proteins with apparent molecular weight of 14, 19, and 25 kd are present also in the HzSO4 extract. The HzSO4 extract is run on an AUT-PAGE where basic proteins are normally separated as histones and protamines (Fig. 4, lane 1).By comparison with the data present in literature (Yokota et al., '91; Mann et al., '82; Kasinsky et al., '85), one can distinguish H3, H4, SP2, SP3-5, and SP6. Electroeluted 14,19, and 25 kd (comingfrom SDS extract) were compared on an AUT-PAGE with an HzSO4 extract; 14 kd migrates as H4 (Fig. 4, lane 2), 19 kd (as expected after amino acid sequenc-
ing) migrates as H3 (Fig. 4, lane 31, and 25 kd migrates as SP2 (Fig. 4, lane 4). Conversely, the H2S04extract (i.e., the extract obtained by the conventional procedure used for basic protein extraction), after a first separation in AUT-PAGE, was run in second dimension in SDS PAGE (Fig. 5).The second dimension shows that histone H4, histone H3, and sperm specific protein SP2 migrates, in SDS-PAGE, as expected for the 14, 19, and 25 kd proteins.
DISCUSSION
Recently, surface proteins of Xenopus spermatozoa which are able to bind to egg envelope components have been identified (Lindsay and Hedrick, '87, '88). We have initiated their characterization as a first step to understand their function. However, these proteins resulted t o be the sperm specific protein SP2 and the histones H3 and H4. Protein 19 kd has been sequenced from Western Blotting and from electroeluted material; both determinations indicate a high identity with histone H3. Moreover, proteins purified from SDS extracts (Lindsay TABLE I . Amino acid sequences of the 19 kdprotein obtained and Hedrick, '88) by electroelution from SDS-PAGE migrate in AUT-PAGE where histones H4 and H3, from blotted (BL) or from electroeluted (EE) material are compared to the sequence of histone H 3 (H3) aspublished by and SP2 of Xenopus are known to migrate. These Old et al. ('85) and Perry et al. ('85) results were confirmed by two-dimensional electrophoresis, where migration patterns due to charge BL A-X-T-K-Q-T-A-X-X-S-T-GEE A-X-T-K-Q-T-A-X-K-S-T-G-G-K-A-P-X-K-Q-L-A-T-K-A-A- (AUT-PAGE)are compared to migration due to mass H3 A-RT-K-Q-T-A-RK-S-T-G-G-K-A-P-RK-Q-L-A-T-K-A-A- (SDS-PAGE).
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Fig. 5 . Two-dimensional PAGE. The acid extract was run for the first dimension in an AUT-PAGE and, for the second dimension, on a 13.5% SDS-PAGE. Arrowheads point to H3, H4, and SP2 that migrate in the second dimension as expected for the 19,14, and 25 kd proteins; 0,origin.
Fig. 4. Proteins coming from an SDS extract and electroeluted from a preparative SDS-PAGE were compared to a sperm acid extract (lane 1)on an AUT-PAGE. Arrowheads of lane 1 point (from top to bottom) to H3, H4, SP2, SP3-5, and SP6. Arrowheads of lane 2 to 4 point, respectively, to electroeluted 14, 19, and 25 kd proteins. The electrophoresis direction was from top ( + ) to bottom ( - ).
Histones and sperm specific basic proteins are known to be localized into the nucleus of mature Xenopus spermatozoa (Moriya and Katagiri, '91). How, then, to explain the experiments of Lindsay and Hedrick ('88)that show that these proteins were 1)surface proteins and 2) able to bind components of the egg coats? The results of the iodination experiments may have suffered from technical problems such as a too harsh iodination; alternatively, we should consider the exiting possibility of nuclear protein exposed t o the external environment. The ability of these proteins to bind to egg coat components is easier to explain as it could be caused by non-specific bindings due to the highly basic nature of these proteins. The discrepanciesbetween the molecular weights of histones H4 and H3 (i.e., 11and 15 kd) and those determined by SDS-PAGE (i.e., 14 and 19 kd) are explained by the exceptional basicity of these proteins and by the consequently reduced overall negative charge. It is well known, in fact, that histones, when analyzed by SDS-PAGE, migrate slower than expected and consequently their molecular weight will be overestimated if compared to non-basic proteins (Hames, '81). Similarly, we ought to forecast a reduced molecular weight for the 25 kd also.
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