THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 1, Issue of January 5, pp. 185-194,1993 Printed in U. S. A.

Sequence and Characterization of a Sperm-specific Histone H1-like Protein of Mytilus californianus”

(Received for publication, April 8, 1992)

Steve Carlos$, Laia Jutglart, Ignacio Borrellv, Donald F. Hunt11 , and Juan Ausio$** From the $Department of Biochemistry and Microbiology, University of Victoriq’Victoria, British Columbia V8 W 3P6, Canada, the sunidad de Quimica Macromolecular, Consejo Superior de Investigaciones Cientificas (CSIC), Escola Tecnica Superior d’Enginyers Industrials de Barcelona Diagonal 647, Barcelona E-08028, Spain, the Wentro de Enserinnza Tecnica Superior Instituto Quimico de Sarria, (Centro Asociado CSIC) Departamento de Quimica Organica, Barcelona E-08017, Spain, and the [[Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901

The major protein fraction of the protamine-like PL- II* (42B) from the sperm of Mytilus californianus has been sequenced and characterized. Immunological and sequence analyses unequivocally show that this protein is indeed a member of the histone H1 family. Along with proteins of the histone H1 class, the protein also shows cross-reactivity and sequence identity, in its NH2-terminal region, with the major protamine-like protein component of Mytilus sperm: PL-I11 (41), of smaller molecular mass. Indeed it is the unusual repet- itive sequence motif of the NH2-terminal domain of PL-11* that bestows to this protein its protamine-like nature. Fourier-transform infrared spectroscopy spec- troscopy indicates that the protein contains consider- able secondary structure: 18% a-helix, 21% &sheet, 39% turns and bends, 22% random coil. At the higher levels of structure, PL-11* exhibits ionic strength-de- pendent folding which is indistinguishable from that of histone H6, as monitored by fluorescence anisotropy.

The chromatin of the sperm of most bivalve mollusks consists of a heterogeneous mixture of protamine-like basic chromosomal proteins (Ausio, 1986). Despite the inter- and intraspecific structural variability of these proteins, they share, in most instances, a strikingly similar amino acid composition (Ausio, 1986, 1988). Because of their amino acid composition these proteins have been considered to belong to a class of proteins intermediate between histones and prota- mines (Bloch, 1969; Subirana et al., 1973). In fact, they were used to establish one of the five major categories in the classification of sperm proteins proposed by Bloch (1969). These proteins define type 3 of this classification also known as Mytilus Type (Bloch, 1969).

At the chromatin level, very little is known about the structures resulting from the interaction of these intermediate sperm proteins with DNA (Ausio and Subirana, 1982a; Ausio and Van Holde, 1987; Zalenskaya et al., 1985; Olivares and Ruiz, 1991). This is in contrast to the more extensive knowl- edge that we have about chromatin organization resulting from histone-DNA interactions (Van Holde, 1989) and to a lesser extent about the structure of the nucleoprotamine complexes (Risley, 1990). Knowledge of the structural details of the sperm intermediate proteins and their relationship to

* This work was supported in part by NSERC Grant OGP0046399 (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** TO whom all correspondence should be addressed.

other well established chromosomal proteins should provide better insight into the structural role played by these proteins in the organization of the sperm chromatin.

In this paper we present the first complete structural char- acterization of one such protein. To fulfill that purpose, we have chosen one of the protamine-like proteins (PL-11* com- ponent) (Ausio, 1986) from the sperm of the organism (My- tilus) which gives the type name to these proteins in Bloch’s classification of the sperm nuclear proteins (Bloch, 1969).

MATERIALS AND METHODS

Living Organisms The specimens of the mussel Mytilus californianus were collected

from Northern Pacific shores (Oregon and Victoria). Sperm collection and the isolation of the sperm nuclear proteins were carried out as described previously (Ausio, 1986).

Protein Fractionation Proteins PL-II*, PL-111, and PL-IV were fractionated from a 0.4 N

HCl extract, using ion-exchange chromatography on CM-Sephadex C-25 as described elsewhere (Ausio, 1986). The first peak eluting from the column consists of all the histones in addition to some oligonucleotides solubilized during the HCl extraction of the nuclear proteins. The proteins from this peak were re-extracted with 5% perchloric acid to solubilize histone H1 and some high molecular weight aggregates of unknown nature. Final purification of H1 was achieved by running this sample through a Bio-Gel P-60 column as described later.

Protein PL-11* consists of a microheterogeneous mixture of pro- teins (Mogensen et al., 1991). The main component of this mixture was fractionated using a C4 (25 X 0.46 cm) 300-A Vydac column eluted at 1 ml/min with a 27-31% linear acetonitrile gradient in 0.1% trifluoroacetic acid over 48 min.

Core histones from the sperm of M. californianus were isolated by selective fractionation following method I1 of Johns (1964). The ethanol/hydrochloric acid fraction, which in this case contained all the histones in addition to some Pl-I11 and PL-IV proteins, was re- extracted with 5% perchloric acid in order to selectively solubilize the PL proteins and histone H1. The resulting perchloric acid-insoluble fraction, containing most of the sperm histones, was then solubilized in 0.4 N HCl and precipitated overnight at 4 “C with 6 volumes of acetone. Further fractionation of these histones was achieved by gel filtration on a Bio-Gel P-60 column according to Van der Westhuyzen et al. (1974) as described elsewhere (Ausio and Van Holde, 1988). Chicken erythrocyte histone H5 was obtained as described elsewhere (Garcia-Ramirez et al., 1990).

Gel Electrophoresis Gel electrophoresis was carried out on (6 M) urea-acetic acid slab

gels prepared according to the method described by Panyium and Chalkley (1969) and modified according to Hurley (1977) as described elsewhere (Jutglar et al., 1990).

185

186 Sperm-specific Histone HI -like Protein of Mytilus

Antibody Preparation

Antibodies to purified PI-11* were obtained from New Zealand White rabbits in the presence of yeast RNA (Muller and van Regen- mortel, 1989). Antibody titers were constantly measured by indirect enzyme-linked immunosorbent assay (ELISA'; protocol to be de- scribed), and only the highest titer was used in the following experi- ments.

Immunoblotting The protein sample from M. californianus was resolved by acid-

urea gel electrophoresis (see above) on a vertical slab gel (24 X 15 X 2 cm). The gel was run at 150 V (-9 mA) until the pyronine dye had reached the bottom of the gel (-4.5 h). At that time, three lanes were sliced from the gel and stained with Coomassie Blue (0.2% Coomassie R-250, 25% isopropyl alcohol, 10% acetic acid) for 30 min, then destained (10% acetic acid, 10% isopropyl alcohol) overnight. The remaining gel was electrophoretically transferred onto nitrocellulose (Shleicher and Schuell Ba 83) using an electrophoretic blotter (Idea Scientific). Transfer of the proteins was performed using 0.7% acetic acid as the buffer and running the transfer at 12 V for 1.5 h. Following transfer, the nitrocellulose was sliced into three distinct pieces each consisting of a single and a double load. One of the pieces was placed in Amido Black stain (0.05% Amido Black, 10% acetic acid, 10% isopropyl alcohol) for 5 min and then destained to determine the efficiency of the transfer. Immunological detection of the proteins was performed as follows. The three remaining slices were washed twice for 5 min in 150 ml of NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 7.4). The slices were then blocked overnight in 150 ml of NET buffer + 1% BSA at 4 "C. After discarding the blocking solution, one of the slices was placed in 100 ml of the first antibody solution (NET buffer + 1% BSA + 0.01% of undiluted anti-phi2B rabbit anti sera) for 1 h at 22 "C with shaking. The last slice was placed in a similar solution except that it contained rabbit prebleed serum to use as an antibody control. The filters were then washed 3 X 15 min with shaking in 150 ml of NET + 0.5% Tween 20 at 22 "C. Next, the filters were immersed in the second antibody solution consisting of NET + 0.5% Tween 20 + goat anti-rabbit IgG peroxidase (Sigma). This was done for 1 h with shaking at room temperature. The antibody solution was then discarded, and the filters were washed 2 X 15 min (with shaking at room temperature) in 150 ml of 10 mM Tris-HC1 (pH 7.3). The last step was to add the freshly prepared dianisidine (Phipps) substrate (0.0025 g of diansidine dissolved in 2.5 ml of methanol was then added to a solution of 0.35 ml of 3% hydrogen peroxide in 200 ml of 10 mM Tris-HC1, pH 7.3) after the bands had developed (-10 min), the reaction was stopped by flushing the filters with distilled water.

ELISA Equimolar solutions of PL-II*, PL-111, PL-IV, histone H2B and

H1 from Mytilus, and histone H5 from chicken erythrocytes were prepared in phosphate-buffered saline (PBS) (0.15 M NaCl, 0.01 M phosphate buffer, 0.01% sodium azide, pH 7.2). 200 pl of each protein solution was pipetted into 24 wells (2 rows X 12 wells) of an ELISA plate (Costar). The first row served as the rabbit prebleed control, while the second row served as a test of cross-reaction with anti-PL- I1 serum. The ELISA plates were incubated overnight at 37 "C. The next day, the antigen solutions were discarded, and the plates were then washed three times with PBS-Tween (PBS -t 0.05% Tween 20). The wells were blocked with 200 pl of a PBS + 3% BSA solution with incubation for 1 h at 37 "C. The wells were then washed three times with PBS-Tween. Serial dilutions of the first antibody solutions (both the anti-PL-11* and the prebleed control) were made in PBS + 0.5% BSA. 100 p1 of each dilution was placed in the appropriate well, and then the plates were incubated for 1 h at 37 "C. The plates were then washed five times with PBS-Tween over 20 min. The second antibody solution (goat-anti-rabbit 1gG peroxidase (Sigma)) was diluted to 1/ 2000 in PBS-Tween + 0.5% BSA. 100 p1 of this solution was added to each well, and the plates were incubated at 37 "C for 1 h. Following that, the plates were washed three times with PBS-Tween over 15 min, and 200 p1 of freshly prepared peroxidase substrate (20 ml of citrate buffer; 50 mM citric acid, pH 4.0, 100 pl of 2,2'-azinobis(3-

The abbreviations used are: ELISA, enzyme-linked immunosor- bent assay; FTIR, Fourier-transform infrared spectroscopy; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HPLC, high- performance liquid chromatography; PL, protamine-like.

ethylbenzthiazolinesulfonic acid) stock; 274.4 mg of 2,2'-azinobis(3- ethylbenzthiazolinesulfonic acid) in 12.5 ml of H20, 80 pl of H202 in 7.5 ml of H2O) was added to each well. The plates were then placed in a drawer away from light for -20 min. 40 pl of stop solution (0.1 M citric acid, pH 4.0, 0.01% sodium azide) was added to each well, and the absorbance of the substrate products were read at 405 nm on an ELISA reader (Dynatech MR5000).

In addition, competitive inhibition ELISAs were undertaken using the above-mentioned protein solutions after determining the opti- mum protein concentration. The competing antigens were incubated with the anti-PL-11* rabbit serum at 4 "C overnight before addition to the coated ELISA plate wells. The remaining steps were identical to the indirect ELISA described above.

Digestion with Proteases Trypsin (EC 3.4.21.4)"Protein PL-11* was digested with trypsin

in the presence of 2 M NaCl, 10 mM (Tris-HC1) pH 7.5 as described before (Ausio et al., 1987).

Endoproteinase Arg-C (EC 3.4.21.40)"Protein PL-11* was dis- solved in distilled water and then brought to 2 M NaCl, 10 mM Tris- HCl, pH 7.5, by addition of an equal volume of 4 M NaCl buffer stock. The resulting solution (A230 E 15) was digested for 120 min at room temperature with arginine endopeptidase from Boehringer Manheim, at an enzyme-to-substrate ratio E / S = 1:lOO (W/W). Afterward the solution was brought to 25% in trichloroacetic acid on ice and centrifuged down in an Eppendorf microcentrifuge at 4 "C for 10 min. After three washings with cold 1 N HC1-acetone, the pellet was dried under low vacuum pressure and finally resuspended in 1 ml of 1 M NaCl 50 mM sodium acetate buffer, pH 6.7, that had been slightly acidified by addition of glacial acetic acid to 5%. The resulting solution was immediately loaded onto a CM-Sephadex C-25 column.

Endoproteinase Glu-C (Protease V-8) (EC 3.4.21.19)"Protein PL- 11* initially dissolved in water (OD 230 = 6 ) was mixed with one volume of 100 mM ammonium acetate pH 4.2. After 10 min at 37 "C, one tenth volume of a glutamic endopeptidase solution at -2 mg/ml, in 50 mM ammonium acetate pH 4.2 was added to that solution and the resulting mixture was incubated at 37 "C for 1 h 50 min, after which the sample was immediately loaded onto a CM-Sephadex C-25 column without any further treatment.

Pepsin (EC 3.4.23.I)"Protein PL-II* (75 A230 in 8 M urea, 5% acetic acid was digested with pepsin at E / S ratio of 1: lOO (w/w) for 20 min at room temperature. The mixture was finally diluted with 10 volumes of 50 mM sodium acetate, pH 6.2, buffer and quickly loaded onto a CM-Sephadex C-25 column.

Chymotrypsin (EC 3.4.21.I)"Protein PL-II* (20 ,4230) in 50 mM Tris-HC1, pH 7.5, was mixed with one volume of 0.02 mg/ml chy- motrypsin with the same buffer, and the mixture was incubated at room temperature for 90 min. The sample was then brought to 3% in acetic acid and loaded directly onto a CS Vydac column.

Carboxypeptidase B (EC 3.4.17.2)-(Boehringer Mannheim and Worthington) digestions were carried out at room temperature in ammonium bicarbonate (100 mM) at pH 8.0 and at 37 "C as described elsewhere (Ausio and McParland, 1989). Different enzyme substrate ratios were used ranging from 1:lOO to 1:5 (w/w).

Carboxypeptidase A (EC 3.4.1 7.1)"Carboxypeptidase A was pur- chased from Boehringer Mannheim and from Worthington. The digestion conditions were identical as for carboxypeptidase B.

Carboxypeptidase Y (EC. 3.4.16.l)-Carboxypeptidase Y from Cal- biochem was used also at different E/S ratios (1:30 and 1:lO). The protein was dissolved in 0.1 M ammonium acetate at pH 5.6 in the presence and in the absence of 1% SDS. When SDS was used, the sample was preincubated at 60 "C for 20 min prior to digestion. Digestions were carried out at room temperature.

Carboxypeptidase P (EC. 3.4.16.I)-Carboxypeptidase P (Boehrin- ger Mannheim) digestions were carried out in 50 mM ammonium acetate at pH 4.2 at E / S = 1:40 to 1:30 (w/w) and at 37 "C. The reaction was stopped either by addition of a few drops of ammonium hydroxide followed by lyophilization or by addition of a one-fifth volume of N,N-diisopropylethylamine in methanol (1:1, v/v) and loaded directly into the amino acid analyzer.

In all the carboxypeptidase digestions L,D-norleucine was used as an internal standard. Aliquots of a buffer solution containing the same amount of enzyme as the sample solution but without protein, were withdrawn simultaneously at the same time intervals as those chosen for the time course of the digestion of the protein (including zero time). In addition to these controls for autodigestion of the carboxypeptidase, a blank control consisting of pure buffer was always

Sperm-specific Histone HI -like Protein of Mytilus 187

analyzed. All the control aliquots were subjected to the same treat- ment as that undergone by the sample prior to being loaded onto the amino acid analyzer.

Chemical Cleavage Cyanogen Bromide Cleauage-Protein PL-11* in 0.1 N HCl was

incubated in the dark and at room temperature in the presence of cyanogen bromide as described elsewhere (Jutglar et al., 1990). The sample obtained was lyophilized, dissolved in 1 M NaCl sodium acetate buffer, and loaded onto a CM-Sephadex C-25 column.

N B S Cleauage-Protein PL-11* was dissolved in 8 M urea, 5% acetic acid at -20 mg/ml. One-fourth volume of N-bromosuccinimide at 20 mg/ml in the same buffer was then added, and the reaction was allowed to proceed for 1 h at room temperature. The reaction was stopped by diluting the reaction mixture with a 10-fold excess of 50 mM sodium acetate buffer (pH 6.7). The solution thus obtained was then immediately loaded onto a CM-Sephadex C-25 column.

Peptide Fractionation Peptide fractionation was routinely carried out on a CM-Sephadex

C-25 column. Elution conditions were as described above for the protein fractionation but with different sodium chloride gradients. In the case of chymotrypsin, peptides were fractionated by reversed- phase HPLC on a Ca (15 X 0.46 cm) Vydac column, eluted with a 0- 30% linear acetonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. In most instances the peptides were purified further using reversed-phase HPLC on a 25 X 0.46 cm, 300-A CIS Vydac column eluted at a flow rate of 0.75 ml/min using different acetonitrile gradients in 0.1% trifluoroacetic acid.

Amino Acid Analysis Amino acid analyses were obtained using an Applied Biosystems

Model 420A derivatizer-analyzer system. The hydrolysis was carried out using gas-phase 6 N HCl and 1% phenol under an argon atmos- phere at 165 "C for 1,2, and 4 h, and the final amino acid composition was obtained by extrapolation of the data to zero time.

Primary Structure Analysis Peptide sequencing was performed on an AB1 Model 470A gas-

phase protein sequenator. The standard AB1 02C ser program was used for coupling and cleavage with the cartridge set at 40 "C. Phenyl- thiohydantoin derivatives were obtained with trifluoroacetic acid at 55 "C. These derivatives were analyzed on a Beckman Microflow HPLC (Downing and Mann, 1976) system equipped with an IBM cyano column, 2-3 nmol of protein/peptide were used in the analysis.

Salt Fractionation of the Sperm Chromatin from Mytilus Sperm chromatin mildly digested with micrococcal nuclease (Wor-

thington) was sequentially extracted with different salts as described elsewhere (Ausio and Van Holde, 1987).

Mass Spectrometry Electrospray ionization spectra were obtained as described else-

where (Hunt et al., 1991).

Sedimentation Equilibrium Sedimentation equilibrium experiments were performed in a Beck-

man Model E analytical ultracentrifuge equipped with interference optics. All the samples were thoroughly dialyzed against 150 mM NaCl, 20 mM Tris-HC1, pH 7.5, before each run. Run conditions were the same as described elsewhere (Ausio, 1988). The partial specific volume of PL-II* was calculated from its amino acid composition (Cohn and Edsall, 1943) and was found to be 0.728 cm3/g.

Fluorescence Measurements Fluorescence anisotropy r = (IIr - IJ/(IrI + 21,) was measured on

a Kontron SFM25 spectofluorimeter as described elsewhere (Ausio, 1988).

FTIR Spectroscopy Fourier-transform infrared spectroscopy (FTIR) was carried out

on a BOMEM Michelson 100, FTIR spectrometer using a high speed deuterated triglycine sulfate detector. All experiments were performed at room temperature (-23 "C). 256 interferograms were collected for each spectrum as in Holloway and Mantsch (1989).

The protein PL-II* and its trypsin-resistant core, first dissolved in DzO and then an equal volume of 2 M NaC1, 100 mM HEPES (pH 7.5) buffer in D,O was added. The final protein concentration was -30 mg/ml (z millimolar). The sample was applied between AgCl windows, using a 50-pm Teflon spacer. Nominal instrument resolu- tion was set at 4 cm". Deconvolution of the spectra was carried out according to Mantsch et al. (1986). Fourier self-deconvolution was performed with BOMEM-CALC FTIR Software (Spectra Calc and Collect Arithmetic C2.12) from Galactic Industries Corp. (1988), which was generously provided to us by MICROBEAM S. A. (Bar- celona, Spain). The deconvolution was carried out with a Bessel apodization function using gamma of 1.23 and a filter of 0.77.

Secondary Structure Prediction

Secondary structure prediction from amino acid sequence was carried out according to Chou and Fasman (1974a, 1974b, 1976) and to Garnier et al. (1978) using a MacVector program (IBI).

RESULTS

Protein PL-11* Is Not a Histone H2B"The nucleus of the sperm of Mytilus contains three major basic proteins: PL-II*, PL-111, and PL-IV (Ausio, 1986) (see Fig. 1). These proteins coexist in the mature sperm with 20-25% of a full histone complement (Ausio, 1986). The smallest one of these highly specific proteins, PL-IV, has already been sequenced (Ausio and McParland, 1989; but see also the accompanying paper (Carlos et al., 1993)).

In M. edulis, the protein with lower electrophoretic mobility PL-11* was known to exhibit an amino acid composition similar to that of somatic histone H2B. However, because of its anomalous solubility in dilute perchloric acid as well as its higher lysine content (Ausio and Subirana, 1982a), an une- quivocal link to histone H2B could never be established despite the initial name of d2B given to this protein (Ausio and Subirana, 1982a).

Table I shows the amino acid analysis composition of the PL-11* protein from M. californianus in comparison to his- tones H2B and H1 isolated from the same sperm cells. As can be seen there, PL-11* shows an intermediate composition between histone H1 and histone H2B. However, when com- pared to the counterparts of these histones from calf thymus, the overall composition of PL-11* looks closer to the compo- sition of histone H2B than t o t ha t of histone H1 (Ausio and Subirana, 1982A), in agreement with the solubility properties of this protein. Indeed, when the method of Johns (1964) is used to fractionate the sperm proteins from Mytilus, PL-11* appears in the organic phase that would selectively solubilize histone H2B from somatic tissues (Subirana et al., 1973) From the point of view of its electrophoretic mobility, PL-11* runs in the region corresponding to histones H2A-H2B (see Fig. 1).

Following the early immunological studies with this protein initiated by Uschewa et al. (1985), which suggested the exist- ence of a relationship between PL-11* and histone H1 but not H2B, we decided to pursue this analysis further. The results obtained, using polyclonal antibodies to PL-II*, are shown in Figs. 1 and 2. Direct ELISA measurements (see Fig. 2), using antiserum elicited against PL-II*, clearly point to the exist- ence of considerable cross-reactivity of PL-11* with histone H1 isolated from the sperm of Mytilus. Significant cross- reactivity is also observed with somatic chicken erythrocyte histone H5 as well as with PL-I11 in the order PL-I11 > H1> H5. No cross-reaction is observed with either sperm histone H2B or PL-IV. Thus i t becomes clear from this simple exper- iment that PL-11* shares no antigenic determinants with histone H2B. Our results corroborate the earlier observations made by Uschewa et al. (1985) using rat liver H2B antibody.

One surprising result that emerged from this immunological

188 Sperm-specific Histone HI-like Protein of Mytilus

A. B. 1.4 - I I ~ 1 2 3 4 5 6

FIG. 1. A , ELISA measurement of the cross-reactions hetween protein PI,- 11’ and the following. I , PL-11’ (0); 2, I’I,-III from Mytilus (a): 3, PL-IV from 1.0 t Mytilus (A); 4, gonadal histone H1 from the sperm of Mytilus (0); 5, gonadal m$o.31 histone H2R from the sperm of M,vtilus (:l>h); 6, histone H5 from chicken eryth- d 0.6 rocvte (9). H, electrophoretic analvsis of the proteins used in A. M , whole HCI 0.4 I rlrotein extract from the nuclei of the sperm of M. cnliforninnus. *‘

TAME I Amino acid composition of some sperm-.sprcifir nuclear proteins frnm M. cnliforninncis in comparison t o thrircnuntcvpnrts in calf thymus

I’I,-lI* HZR-MC” H I - M C HZR-CT” H1-CT’

mol% Lys 18.4 12.2 29.1 14.1 26.8 His 0.6 x n 0 5 Arg

2.3 9.2 8.6 .5.7 6.9 1 .8

Asx 5.2 6.0 4.8 5.0 Thr 4.2 I ..) 4.9 6.4 5.6

2.5

Ser 16.7 7.8 10.7 10.4 5.6 Glx 1.5 7.9 4.0 8.7 3.7 Pro 7.9 4.2 7.1 4.9 9.2 GlV 8.1 1 . 1 5.2 5.9 7.2 Ala 1 3 . 3 10.8 12.6 1n.8 24.3 112 c y s t rh t r Val 3.5 6.6 4.3 1 . I ) 5.4 Met 2.0 1 .9 1.7 1.5 Ile 3 . 0 5.4 3.6 5.1 1.5 Leu 4.5 6.0 4.4 4.9 4 3 Ty r 0.5 2.0 0.4 4.0 0.9 I’he 1.4 2.2 1 .o 1.6 0.9

- r

”

- r

“The ahhreviations used are: MC, M. cnlifnrnianus; CT, calf t hvmus.

tr, trace. From Maves and Johns (1982).

A B C D 1 2 i - 1 2 1 2 2 1

PI -

PL-I

PL- IV

L

ANTISERUM DILUTION

characterization was the high level of Cross-reactivity ob- served between PL-11’ and P L - 1 1 1 . Immunoblotting analysis allowed us to explore this further (see Fig. 2 ) . A s can be seen in this figure, of the three PI, components present in Myti1u.s sperm, anti-PL-11’ specifically recognizes PL-11’ and cross- reacts to a lesser extent with PI,-111. No cross-reactivity at all was observed with PL-IV.

The finding of a cross-reaction between some of the PI , components disagrees, however, with previous immunological characterization studies (Uschewa rt al., 1985) carried out with protein PL-11’.

The True Histonr HI Naturt. of I~I,-II*-As has been men- tioned in the preceding section, PL-II* exhibits considerable cross-reaction with the histones of the HI family. This finding was not surprising, considering that PI, proteins with histone HI-like features have previouslv been found in other bivalve mollusks (Giancotti rt a/., 198.7: Ausio rt 01.. 1987: Ausio, 1988). We are going to show next that in fact PI,-11’ meets all the criteria reqiured to he considered a true sperm-specific histone H1 variant. A s described earlier (Ausio and Suhirana. 1982), PL-11’ is soluble in 5‘; perchloric acid. Of a11 the histones, only histone H1 members are soluble under thew conditions. In addition, PL-11’ is extracted from chromatin at about 0.6 M NaCl like histone H1. This is at a much lower ionic strength than that required to extract any other kind of histone or protamine.

Fig. 3A shows the solubility o f the chromatin of M . califor- nianus as a function of the ionic strength. Also shown in Fig. 37R are the proteins extracted at 0.6 M NaCI. As i t can he clearly seen in this figure, PL-11’ and PI,-IV are extracted together at 0.6 M NaCI, much before any considerahle amount of DNA is solubilized. Indeed, like PI,-11’. P L - I V has also been related to the lysine-rich histone H1 (Phelan rt 01.. 1974). The chromatin fraction not extracted with 0.fi M NaCI contains 2 90rA of the DNA a s well as the core histones (H.7, H2R, H2A, and H4) and PL-111. This later PI, component which amounts to approximately 70‘; of the whole P I , nuclear contents of Mytilu.7 sperm (Ausio and Subirana, 1982) is most likely responsible for the insolubility of chromatin upon re-

which was use> as a tracking dve. . .

found that under the salt extraction conditions used by us.

A. B.

PL-II'

PL-Ill

PL-IV

NsCI. IMI

FIG. 3. A , solubilization o f Mvfilus sperm chromatin as a function o f the ionic strength. Oprn and hlncl; dots represent two independent sets of experiments. Also shown as a comparison are the solubilization I)rofilcs, ohtained under similar conditions with: (doshrd lints) Spi- sula so/idissimn (surfclam) (Ausio and Van Holde. 1987). and (dottrd l inr s ) mouse liver (Sanders. 1978). H . urea-acetic acid gel electropho- resis of M, total sperm nuclear proteins from M. cnlifornianus: I , proteins associated with the 0.6 M NaCl soluble fraction; 2, proteins remaining associated with the insoluhle chromatin fraction, under the same rondit ions.

PL-IV is always solubilized t.ogether with PL-II* in agreement with an earlier report (Zalensky et al., 1982). Some of this disagreement could he accounted for by the different experi- mental conditions used, especially hecause of the use of a mild nuclease digestion previous to the salt extraction and the lower temperatures (4 "C) employed by us.

A more stringent requirement for a chromosomal protein to be considered a real histone H1 is the presence of a n internal glohular trypsin-resistant core (Allan et of., 1980) which in turn corresponds to the most sequence-conserved par t of H I histone (Cole, 1984). We therefore isolated histones H1 and H2H from the histone fraction of sperm nuclear proteins. PL-lI*, H2R, and H1 were then digested with tr-ypsin under identical conditions. The results of such digestions indicated that histones HI and PL-II* produce tr-ypsin-resist- ant cores which exhibits an almost identical mobility in urea- acetic acid gel electrophoresis. In contrast, histone H2R yields a heterogeneous mixture of peptides with completely different electrophoret,ic behavior (results now shown). The trypsin- resistant core of PL-11' has been shown to contain 84 amino acids, and its primary structure exhibits a high degree of similarity with the cores of other histone H1 proteins (.Jutglar et al., 1990).

The structural relatedness of PL-11* and histone HI is also supported by the fractional change in folding as a function of ionic st,rength measured by fluorescence anisotropy. These data indicated identical salt-dependent folding of PL-II* and chicken erythrocyte histone H5 (results not shown). The use of histone H5 for comparison in this case, was based on the following criteria: 1) T h e globular cores of histone H5 and PI,-II* exhihit a high degree of sequence similarity (dutglar et af., 1991); 2) Both proteins belong to a terminally differ- entiated cell system; 3 ) Histone H.5 is one of the smallest somatic H1 histones and therefore should compare better to PL-II*. As mentioned above the salt-dependent folding of histones H5 and PI,-II* is virtually identical, with a midpoint transition (at -120 mM NaCI) which is verv close to the values reported for other histone H1 proteins (Smerdon and Isenberg, 1976).

Primary Structurc of PL-II*-We have recentlv shown that the protein PI,-II* from Mvtilus consists of a microheteroge- neous mixture of peptides with interspecific variabilitv (Mo- gensen ct nl., 1991).

An initial analysis of the molecular mass o f the unfraction- ated PL-lI* protein bv sedimentation equilihrium, using in- terference optics and the meniscus depletion method (Yphan- tis, 1964), revealed a value of 15.300 t 501) I)a corresponding to approximately 14.1 2 -5 amino acids.

The major protein component of I' lA-l l* I representing ahout 70% of the mixture) was fractionated hy reversed-phast. HPLC. Mass spectrometry of this fraction provided a molec- ular mass of 15,601 t 2 for this fraction which, despite the HPLC purification, was still found to contain a small amount of a residual component of molecular mass 15.865 t I O . The whole sequence of PL-II* as well as the strategy fnllnweti to provide the necessary overlapping peptides is shown in Fig. 4A. The purity of some of the peptides used for this analysis is shown in Fig. 4H. Tatde I1 also shows the amino arid yields for the NH,-terminal (P-2) pepsin peptide and for the ('OOH- terminal chymotr-ypsin peptide (CH).

The presence of alternative amino acids at different posi- tions of the sequence (see Fig. 4A ) is indicative of some further microheterogeneity. Indeed, protein microheterogeneity is a well-documented feature of proteins of the histone H1 family (Cole, 1984).

One of the major problems encountered in determining the sequence of PL-11' was establishing the last few ('OOH- terminal residues o f t h e molecule. A first attempt at comhin- ing carboxypeptidases A and R in a similar approach to that described elsewhere (Ausio and McParland. 1989) turned o u t to be completely unsuccessful. Seit her carboxypeptidase A nor R o r Y cleaved PL-II* at all. At the highest enzyme to substrate ratios that we tried f 1 5 f E/S, w / w ) ) all these enzymes exhibited a high nonspecific endopeptidase activity (most likely from contamination hy other enzymes) which resulted in extensive degradation of the peptide as assessed by electrophoresis. The activity of each of the carboxypepti- dases used was routinely checked with a positive control previous to their use with PI,-II*. P L I Y from M. cnliforninnus was used as a positive control (Phelan P I 01.. 1974; Ausio and McParland, 1989). Of the carhoxbpeptidases tested only car- boxypeptidase P released significant amounts of COOH-ter- minal amino acids. However the results obtained with this enzyme turned out to he quite unreliable due to contaminating endoproteinase activity present at the enzyme-to-suhstrate ratios required for amino acid release.

We therefore decided to use a completely different ap- proach, hased on the amino acid composition of the COOH- terminal chymotryptic peptide CH (see Table 11) and on the molecular mass of the COOH-terminal cyanogen hromirie peptide. Table 11 shows a comparison between the number of amino acids expected from the amino acid composition of CH and the amino acids observed from the sequence. Hoth values are in verv good agreement. In addition to this. the molecular mass of the COOH-terminal cyanogen peptide that overlaps with this chymotryptic peptide was also measured by elec- trospray ionization (see Fig. 5). The value fnund 6351 t 2 1)a (see Table 111) compares very well with the mass of 6372 calculated from the main sequence of that peptide. The small discrepancy found hetween these two values most likelv re- flects the extent of microheterogeneity ohsened within this region (see Fig. 4A and Fig. 5 ) and/or some slight chemical modification introduced t y cyanogen hromide.

All this clearly supports NKSSN being the end o f the sequence for the major protein fraction of P L - I l * of the sperm of M . cnlifornianus (see also accompanying paper Carlos r~ nl. (1993)). Furthermore, the molecular masscalculated from this sequence, 15,602 Da. coincides with that o f I5fOl '. 2 1)a determined by mass spectrometry.

190 Sperm-spwific Histone HI -like Protein of Myti1u.s

A B 1 2 3 4

IT1 I K I 10

b S P S R R S R S R S R S R S K S P K R S P A K K A R K I P K K R R A T G G A K xJK1 30 IKI 40

., IT1 5 0 IN) 80 70 110 K P S T L S M I V A A I Q A M K N R K G S S V Q A I R K Y I L A N N K G I N I S

CNB R- E

TRP

8, PO 10-2 110 110

-NBS - R L G S A M K L A F A K G L K S G V L V R P K T S A G A S G A T G S F R V G K A

E-2 V-8

- NBS - P-3 - - CH-

txlAl 130 (PI 1.0 IKI IN1 P S S P K K K A K K A K S P K K K S S K K S S N K S N N - - _ _ ~ ~ _ ~ p-3 ~ -

C I

10

P S P T R R S K S R S K S R S R S R S A S A G K A A K R A K S K T A K 70 IRi ~ R I 30 (GI

IRI

5 6 7 8 9 M

I

II

II

V

FIG. 4. A , complete amino acid sequence of PI,-II* showing some of the peptides used in the analysis. WP, whole protein; CH. COOH- terminal chymotrypsin peptide; (,'NH, cyanogen hromide peptide; 17-2. peptide ohtained by digestion with elastase; 'VHS. .V-hromosucciniimide peptide; /J-2, /J-.'j, pepsin-generated peptides: If-I.:, peptide ohtained hv endoproteinase Arg-C; V - 8 , peptide ohtained by .vtnphyhm-rus nurrus V-X protease (endoproteinase Glu-C); I ] . electrophoretic analvsis in urea-acetic acid gel of some of the peptide fragments used. I . E-2; 9. P-3: ./, lJ-2: 6 , V-X ; 8 , NHS. Also shown are the starting mixture of peptides generated by the following. 2. elastase; 5 , pepsin: i, Staphylococcus nurcus V - X protense; 9, N-hromosuccinimide cleavage. M is a mixture ofproteins used as a marker and consisting of I , 1'1,-I from S'. solidissimn ( M , :K!,500 I h ) (Ausio and Suhirana, 1982h); 111, 1'1,-I11 from M. edulis ( M , 9,600 Da) (Ausio and Subirana, 1 9 x 2 ~ ) ; I V , Cluprinr ( M , -4,500 I k ) . C', partial sequence from the NH2-terminal region of P I A - I 1 1 from M . californianus.

Drterminntion of the Secondary Structuw Pl-II' Using F'I'IR-In recent years, FTIR has become one of the most powerful tools used to determine secondary structure of pro- teins in solution (Susi, 1972; Koenig and Tahb, 1980; Surewicz and Mantsch, 1988). When careful precautions are taken, especially for the suhtraction of the artifactual absorption hands due to liquid and gaseous water (Dong ~t al., 1990), it is possihle t.o deconvolute the resulting spectrum into its constitutive frequencies (Mantsch et al., 1986). Highly accu- rate qualitative and quantitat.ive information on the second- ary structure of a protein can he obtained by further analysis of these frequencies.

In Fig. 6, we show the deconvoluted spectrum of the amide I region of P I - I I * . The frequencies observed and their second- ary structure assignment was made according to Ryler and Susi (1986): 1680 cm", turns and bends; 1670 cm", turns and bends; 1661 cm", turns and bends; 1651 cm", cr-helix; 1641 cm" random coil, 1632 cm-I, [j sheet, 1622 cm-l, [3 sheet). The quantitative data obtained from this analysis are pre- sented in Tahle IV. These data agree reasonably well with the values predicted by analysis using different algorithms (Chou and Fasman, 1974a, 1974h, 1976; Gamier e t al., 1978). As in t h e case of the sperm-specific protein PL-I from Spisula solidissirno the values predicted for n-helix are higher than those experimentally measured, whereas those opposite are also true for the /$sheet assignment.

Although [,'-sheet folding went undetected in the early struc- tural analysis of histone H1 using circular dichroism (Rrad- Iwry rt al., 1975; Smerdonand Isenberg, 1976), there seems to be reasonable evidence for the presence of this kind of struc- ture in this protein (Pepe et al., 1990). Sequence regions with &sheet forming potential have been described in the NH,- and COOH-terminal regions of the sperm-specific histone H1

from sea urchins (Hill et al., 1989). Thus, it is not surprising to find the presence of both n-helix and +sheet in the prot- amine like (PL-II*, PL-I) sperm-specific H1 histones from bivalve mollusks.

DISCUSSION

We have isolated and characterized the protein PL-11' from the sperm of M. californianus. Together with two other PI, proteins (PL-I11 and PL-IV) this protein coexists in the nucleus of the sperm with -20% of a full complement of somatic-like histones including histone H1.

T h e molecular mass of this protein determined by mass spectrometry was 15,601 Da. This value is consistent with its electrophoretic mobility (between histones H2R and H2A) in urea acetic acid gels (see Fig. 1).

Its solubility in dilute perchloric acid (5r; perchloric acid) as well as the ionic strength (-0.6 M NaCI) at which the protein can he selectively dissociated from chromatin sug- gested a chemical and functional relationship of this protein with histone HI. Indeed, immunological characterization of this molecule (see Figs. 1 and 2) clearly shows that PI,-11' is more closely related to the proteins of the histone H1 family than to any other proteins including histone H2H. The onlv exception to this was its high cross-reactivity with PL-111 which is the major protein component of Mytilus sperm. In fact, the immunological cross-reactivity between PL-II* and PL-111 was the highest of all the proteins analyzed. Thus these proteins must have some common inmmunogenic de- terminants. As can he seen in Fig. 4'2, partial sequence analy- sis of PL-111 shows that there is sequence identity over the first 17 amino acids of the NH,-t,erminal regions of these two proteins. In fact, when the Hopp and Woods algorithm (Hopp and Woods, 1981) was used to predict and identify the loca-

Sperm-specific Histone HI -like Protein of Mytilus 191

TABLE I1 Sequencing yields for the NH2-terminal pepsin peptide P-2, COOH-terminal chymotrypsin peptide CH, and amino acid composition

The values of the yields shown have been corrected for the background. Residues 25 and 31 of CH peptide were assigned from the raw data.

P-2 CH Amino Compositionm Expected compositionb Observed composition'

Residues Yield Residue Yield acid (no amino acids) (no amino acids)

1 P 2 S 3 P 4 S/T 5 R 6 R 7 S 8 R/K 9 s

10 R 11 S 12 R 13 S 14 R 15 S 16 K 17 S 18 P 19 K 20 R 21 S 22 P 23 A 24 K 25 K 26 A 27 R 28 K 29 T 30 P 31 K

prnol 3730 4280 2700 2500/950 1060 1440 2300 500/500

1440 480

1060 325 670 200 150 440 200 180 100 200 70 50

105 50 30 30 23 10 30 15 8

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

R V G K A P S/A S P K K K A K K A K S P K K K S S K K S SIK N K/N S N N

prnol 1191 1699 1356 1531 1860 1189 457/240 332 545 715 787 93 1 740 874 730 732 561 177 312 156 462/138 170 108 47

79 33

63 40/14

32 18

2.51-

% rnol/rnol K 42.2

R 3.0

N N

S 19.0

P 9.3

G 3.7

A 10.3

V 3.0

14

1

3

6-7

3-4

1

3-4

1

13-14

1

3

6-7

3-4

1

3-4

1

Amino acid analysis values corrected for hydrolytic losses. * The values in this column correspond to the number of amino acids calculated from (a) on the assumption that the peptide contains only

E Number of amino acids calculated from the sequencing data shown. 1 glycine and 1 valine residue.

tions of antigenic determinants along the PL-11* protein sequence, this NHz-terminal sequence was found to be the region with the highest antigenic potential (see Fig. 7B). However, it is known that, in the case of histones, inclusion of RNA in the immunogen enhances the antibody specificity for the NH2- and COOH-terminal regions of these molecules (Shay et al., 1988).

This observation provides additional evidence for the exist- ence of a link between these proteins at the protein synthesis level (Bloch, 1969). Yet, it does not support the kind of product-precursor relationship initially postulated (Bloch, 1969). The presence of common peptides in PL-11* and PL- 111, especially in their NHz-terminal regions suggests a pos- sible linkage at the level of the expression and/or organization of their genes. The simplest hypothesis would be to assume tha t these genes share a common exon. This possibility is now being investigated in our laboratory.

That protein PL-11* is indeed a true histone H1 is clearly corroborated by the primary structure of its major component. In an exhaustive comparative search carried out between the sequence of PL-11* and 21512 protein sequences available from the N brf-SWISS data base, we found that the higher identity scores belonged to the proteins of the histone H1 family. Thus, the best score found was a 37.5% identity in a

136-amino acid overlap for the gonadal histone H1 from the sea urchin Parechinus angulosus (Strickland et al., 1980). Following that were: gonadal histone H1 from another sea urchin Echinolanysus crussa (Strickland et al., 1982) (38.2% identity in a 132-amino acid overlap); sperm histones H l a and Hlb from Platynereis dumerilii (annelid) (Kmiecik et al., 1985) (39% identity in a 105-amino acid overlap); embryonic histone H1-P from Strongylocentrotus purpuratus (purple sea urchin) (Lai and Childs, 1988) (40% identity in a 115-amino acid overlap), to give a few examples. In positions 13 and 15 of this comparative analysis is histone H5 from goose eryth- rocytes (Anser anser) (Yaguchi et al., 1976) and histone H5 from chicken (Gallusgallus) (Briand et al., 1980). A significant identity was also found with protamines, especially with gal- line from Gallus gallus (Nakano et al., 1976), mugiline from Mugiljaponicus (Okamoto et al., 1987), and iridine from Salmo irideus (Ando and Watanabe, 1969). The similarity in all these instances is always found to occur within the NH2- terminal region of PL-II*. This provides support for the protamine-like (PL) nature of the PL-11* protein. No histone H2B was found within the range of protein sequences ana- lyzed (50 from the nucleotide sequence bank and 50 from the protein sequence bank) down to values of 18-20% identity.

Further sequence support for the histone H1 nature of PL-

Sperm-specific Histone HI -like Protein of Mytilus 192

100

80

n 0-

60 m U

2 > .- L - d 41

2

I [M+12H112+ 47 55 86 ?C

(M+llH]"+

[M+lOHjlo+

I [M+gH19i

400 600 800 1000 1200 1400

mb FIG. 5. Electrospray ionization spectrum of the COOH-ter-

minal cyanogen bromide peptide of PL-II*. The spectrum was obtained from approximately 5 pmol of peptide.

TABLE I11 Calculated mass from the [M + HI+ ions observed i n the ESI mass

spectrum of the COOH-terminal cyanogen bromide fragment (residues 87-146) of PL-IIY

Charge Observed m/z Calculated mass

+13 490 6358 +12 530.5 6355 +11 578.7 6356 +10 536.8 6359

+9 707.4 6359 +8 795.6 6358

11* comes from the analysis of its trypsin-resistant core. The primary structure of this peptide, not only retains some of the conserved sequences within this histone domain, but also its consensus sequence (Jutglar et al., 1991). The trypsin-resist- ant core represents the most highly conserved sequence do- main in the histones of the H1 family. As it can be seen in Fig. 7B, PL-11* also contains three sites of high antigenicity potential, which are most likely responsible for the cross- reaction of this protein with histone H1 from Mytilus and histone H5 from chicken (see Fig. 1). The main component of protein PL-11* contains 148 amino acids in good agreement with its molecular mass determined by mass spectrometry. This number is relatively low when compared to the amino acid content of some of the most typical representatives of the histone H1 family: 220 amino acids for calf thymus histone H1 and 205 amino acids for chicken erythrocyte histone H5. Yet it compares with sperm-specific histone H1 from other marine invertebrates, e.g. histones Hla and Hlb from the annelid Platynereis dumerilii (Kmiecik et al., 1985). Never- theless, PL-11* has the smallest COOH-terminal domain ever found in an H1 histone (26 amino acids). The COOH-terminal region of histone H1 is involved in the interaction with linker DNA in somatic chromatin and in sperm chromatin it has

I I I I I

I I I I I 1700 1680. 1660 1640 1620 1

WAVELENGTH, cm" 00

FIG. 6. Deconvolution (solid line) and second derivative (dashed line) of the amide I band of the infrared spectrum of PL-11* obtained by FTlR analysis.

TABLE IV Secondary structure analysis (%) of the PL-IF protein in comparison

to PL-I from the sperm of S. solidissima PL-II* PL-I

FTIRe Sequence CDc Sequence analvsisb analvsisb

a-Helix 18 22 5 27 ,%Sheet 21 13 15 11 Turns and bends 39 7d 46 62 Random coil 22 58 39 Calculated from the relative intensities of the peaks of the amide

I region. Prediction according to Chou and Fasman (1974) and Garnier et

al. (1978). The percentages shown correspond to the overlapping regions, predicted by both methods.

e Circular dichroism analysis of PL-I from S. solidissirnu (Ausio et al., 1987).

This value only corresponds to turns.

been suggested to be responsible for bringing together differ- ent DNA regions of chromatin (Subirana, 1990). Thus, the reduction in the number of amino acids in the COOH-terminal domain of PL-11* seems quite surprising in view of its impor- tant role in chromatin and DNA condensation. At this point it is interesting to consider the possibility that PL-11* may operate in conjunction with PL-IV in accomplishing its func- tional role. Indeed, PL-11* and PL-IV are simultaneously coextracted in 0.6 M NaCl (see Fig. 3B). In addition, P1-11* has been shown to account for -20% of the total nuclear protein and PL-IV for -6% (Avramova et al., 1984) which when taken together with the molecular masses of these proteins (15,600 Da for PL-11* and 6,500 Da for PI-IV (Carlos et at., 1993), it shows that PL-11* and PL-IV are present in stoichiometric amounts in the sperm nuclei. Furthermore, PL-IV can be compositionally related to the COOH-terminal regions of histone H1 (Phelan et al., 1974). Indeed the se- quence of the COOH terminus of PL-11* and PL-IV (Ausio

Sperm-specific Histone HI -like Protein of Mytilus 193

FIG. 7. A, hydrophilicity; B, antigenic Index; C, secondary structure prediction for protein PL-11* as a function of the amino acid number. The algorithms used in the analysis were as follows. A, Kyte and Doolitle (1982); B, Hopp and Woods (1981); C, Chou and Fasman (1974a, 1974b, 1976) and Garnier et al. (1978); D, Schematic representation of the structural domains of protein PL-II*.

A.

B.

C.

c. nn . _I. "Y

4 . 0 0 ! I I I I I I .. . ~

c 3.00

1.00 .- m

.r 0 2.00

e -1.00

- 5 0.00

w -2.00 I -3.00 1 -4.00 I

2 I I - I I I I I 1 I I 1 I I 1

-2. "" 1 I I I 20 40 60 80 100 120 140

1.00 0.80

8 0.60 c 0.40 " 0.20 'E 0.00 8 -0.20

V -

z -0.40 5 -0.60 -0.80 -1.00

20 40 60 80 100 120 140

D.

- 34A - and McParland, 1989) seems to account for the structural changes that have been shown to be associated in the transi- tion to sperm histones (Subirana, 1989). An a-helical config- uration has been observed in the COOH-terminal domain (Hill et al., 1989) similar to what has been found in PL-11* (see Fig. 7C) and also in PL-IV (Ausio and McParland, 1989).

In this regard, we note that a structural motif, similar to the "SP-Basic-Basic" repetitive unit described in histones from the sperm of other marine invertebrates (Von Holt et al., 1983), is also observed within the NHp-terminal region of PL-11* (see Fig. 4A). In PL-II*, a simpler "S-Basic-S" version is additionally present (see Fig. 4A). As with SPXK/R in sea urchins (Hill et al., 1990), this repetitive structure may play a very important role in the modulation of histone-DNA inter- actions, either during spermiogenesis or during the events that precede oocyte fecundation. Furthermore, distinctive sec- ondary structure different from a-helix or @-sheet has recently been proposed that allows the SPKK motif to be considered as a new nucleic acid-binding structure in proteins (Suzuki, 1989). It consists of compact @-turns stabilized by an addi- tional hydrogen bond. This structure might could account for the discrepancy found here between the extent of @-turns observed experimentally and the predicted values (see Table IV). Indeed, when only the Chou and Fasman (Chou and Fasman, 1974a, 197413, 1976), algorithm is used for the sec- ondary structure prediction, a long stretch of @-turns is pre- dicted in the NHp-terminal region of PL-11* (see Fig. 7C).

At higher levels of structure PL-II*, like histone H1, con- sists of three well defined structural domains. As can be seen in Fig. 7A (see also Fig. 70 ) , a central globular core is flanked by two extended hydrophilic domains (Jutglar et al., 1991). Thus, while PL-11* maintains most of the basic structural features of the protein members of the histone H1 family, it

provides another striking example of the extent of heteroge- neity within this protein family (Cole, 1984).

Acknowledgments-We thank Janice Hunt and LeAnn Howe for carefully reading the manuscript and Cheryl Gonnason for her skillful typing.

Addendum-While this paper was being reviewed the partial se- quence of the NH,-terminal regions of EM1 and EM6 from Ensis minor were published (Giancotti, V., Buratti, E., Santucci, A., Neri, P., and Crane-Robinson, C. (1992) Biochem. Biophys. Acta 1119, 296-302). The primary structure of these regions exhibits a dipeptide repeat SR(K) which is very similar to that reported in this paper for the NHz-terminal regions of proteins PL:II* and PL-111.

REFERENCES Allan, J. Hartman, P. G., Crane-Robinson, C., and Aviles, F. S. (1980) Nature

Ando, T., and Watanabe, S. (1969) Int. J . Protein Res. 1, 221-224 Ausio. J. (1986) Como. Biochem. Phvsiol. 85B. 439-449

288,675-679

Ausio; J. (1988) J. Bbl Chem. 263,"10141-10150

Ausio, J., and McParland, R. (1989) Eur. J. Biochem. 182,569-576 Ausio, J., and Subirana, J. A. (1982a) Exp. Cell Res. 141 , 39-45

Ausio, J., and van Holde, K. E. (1987) Eur. J . Biochem. 1654,363-371 Ausio, J., and van Holde, K. E. (1988) Cell Differ. 23,175-190 Ausio, J., and Subirana, J. A. (1982b) J. Biol. Chem. 2 5 7 , 2802-2805 Ausio, J., and Subirana, J. A. (1982~) Biochemistry 2 1 , 5910-5918 Ausio, J., Toumadje, A., McParland, R., Becker, R. R., Johnson, W. C., Jr., and

Avramova, Z., Zalensky, A,, and Tsanev, R. (1984) Exp. Cell Res. 152,231-239 Bloch, D. P. (1969) Genetics 6 1 , (suppl.) 93-111 Bradbury, E. M., Cary, P. D., Chapman, G. E., Crane-Robinson, C., Danby, S.

E., Rattle, H. W. E., Boublik, M., Palau, J., and Aviles, F. X. (1975) Eur. J. Biochem. 62,605-613

Briand, G., Kmiecik, D., Sautiere, P., Wouters, D., Borie-Loy, O., Biserte, G., Mazen, A,, and Champagne, M. (1980) FEES Lett. 112,147-151

Byler, M. D., and Susi, H. (1986) Biopolymers 2 5 , 469-487

Chou, P. Y., and Fasman, G. D. (1974a) Emhemutry 13,211-222 Carlos, S., Hunt, D. F., and A d o , J. (1993) J. Biol Chem. 268,195-199

Chou, P. Y., and Fasman, G. D. (1974b) Biochemistry 13,223-245 Chou, P. Y., and Fasman, G. D. (1976) Adu. Enzymol. Relat. Areas Mol Biol.

van Holde, K. G. (1987) Biochemistry 126,975-982

A?. d5-ldR Cohn, E. J., and Edsall, J. T. (1943) Proteins Amino Acids and Peptides, Van

Cole, R. D. (1984) Anal. Biochem. 136,24-30

", _- __- Nostrnad-Reinhold, Princeton, NJ

194 Sperm-specific Histone HI -like Protein of Mytilus Dong, A,, Huang, P., and Caughey, W. S. (1990) Biochemistry 29,3303-3308 Downing, M. R., and Mann, K. G . (1976) Anal. Biochem. 74,298-319 Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120,97-120 Giancotti, V., Russo, E., Gasparini, M., Serrano, D., Del Piero, D., Thorne, W.

A., Cary, P. D., and Crane-Robinson, C. (1983) Eur. J. Biochem. 136 , 509- 516

Garcia-Ramirez, M., Leuha, S., and Ausio, J. (1990) Prot. Ex. Pur. 1 , 40-44 Hill, C. S., Martin, S. R., and Thomas, J. 0. (1989) EMBO J. 8,2591-2599 Hill, C. S., Packman, C. L., and Thomas, J. 0. (1990) EMBO J. 9,805-813

Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,3824- Holloway, P. W., and Mantsch, H. H. (1989) Biochemistry 28,931-935

3828 Hunt, D. F., Alexander, J. E., McCormick, A. L., Martino, P. A., Nichol, H.,

Shabanowitz, J., Sherman, N., Masely, M. A,, Jorgenson, J. W., and Tomer,

2, pp. 441-454, Academic Press, NY K. B. (1991) in Techniques in Protein Chemistry (Villafranco, J. J., ed) Vol.

Hurley, C. K. (1977) A d . Biochem. 80,624-626 Johns, E. W. (1964) Biochern. J. 92,55-59 Jutglar, L., Borrell, I., and Ausio, J. (1991) J. Biol. Chem. 2 6 6 , 8184-8191 Kmiecik, D., Sellos, D., Belaiche, D., and Sautiere, D. (1985) Eur. J. Biochem.

Koenig, J. K., and Tabh, D. L. (1980) in Analytical Applications of FT-IR to 160,359-370

Molecular and Biological Systems (Durig, J. R., ed) pp. 241-255, D. Reidel, Boston

Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157 , 105-132 Lai, C. Z., and Childs, G . (1988) Mol. Cell. Biol. 8, 1842-1844 Mantsch, H. H., Casal, H. L., and Jones, R. N. (1986) in Spectroscopy of

Biological Systems (Clark, R. J. H., and Hester, R. E., eds) pp. 1-46, Wiley,

Mayes, E. L. V., and Johns, E. W. (1982) in The HMG Chromosomal Proteins New York

Mogensen, C., Carlos, S., and Ausio, J. (1991) FEBS Lett. 282 , 273-276 (Johns, E. W., ed) pp. 223-247, Academic Press, New York

Muller, S., and Van Fkgenmortel, M. H. V (1989) Methods Enzymol. 170,251-

Nakano, M., Tohita, T., and Ando, T. (1976) Int. J. Pept. Protein. Res. 8,565- 263

578

Okamoto, Y., Muta, E., and Ota, S. (1987) J. Biochem. 101 , 1017-1024 Olivares, C., and Ruiz, S. (1991) Mol. Cell. Biochem. 101,93-99 Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130,337-346 Pepe, I., Catasti, P., Rauch, G., Nizzari, M., and Nicolini, C. (1990) Biochim.

Phelan, J. J., Colom, J., Cozcolluela, C., Subirana, J. A,, and Cole, R. D. (1974) Biophys. Acta 1041 , 14-21

Risley, M. (1990) Chromatin Organization in Sperm in Chromsomes: Eukaryotic, J. Biol. Chem. 249,1099-1102

Prokarytic and Viral (Adolph, K., ed) pp. 61-85, Vol. 11, CRC Press, Boca

Sanders, M. (1978) J. Cell Biol. 79,97-109 Shay, C. E., Foster, P. G., and Neelin, J. M. (1988) Comp. Biochem. Physiol.

Smerdon, M. J., and Isenherg, I. (1976) Biochemistry 15,4233-4242 Strickland. W. N.. Strickland, M.. and Von-Holt. C. (1982) Biochem. Biophys.

Raton, FL

9 1 B , 69-78

Acta 700,127-iZ9

and Wittmann-Liebold, B. (1980) Eur. J. Biochem. 104,567-578

. .

Strickland, W. N., Strickland, M., Brandt, We. F., Von-Holt, C., Lehmann, A,,

Suhirana, J. A. (1990) Biopolyurers 2 9 , 1351-1357 Suhriana, J. A,, Cozcolluela, C., Palau, J., and Unzeta, M. (1973) Biochem.

Surewicz, W. K., and Mantsch, H. H. (1988) Biochem. Biophys. Acta 962,115- Biophys. Acta 317,364-379

12n Suii,-H. J. (1972) Methods Enzymol. 26,445-472

Uschewa, A,, Patriotis, C., and Avramova, 2. (1985) Cell Biol. Int. Rep. 9 , 253- Suzuki, M. (1989) EMBO J. 8, 797-804

3G2 Van Holde, K. E. (1989) Chromatin, Springer-Verlag, New York Van der Westhuyzen, D. R., Bohm, E. L., and Von-Holt, C. (1974) Biochem.

Yaguchl, M., Roy, C., and Sehgy, V. L. (1979) Biochem. Biophys. Res. Commun.

-"

Biophys. Acta 359,341-345

90. 1 401)-I 406 Yphantis, D. A. (1964) Biochemistry 13 , 297-317 Zalenskaya, I. A,, Odintsova, A. N., and Vorob'ev, V. I. (1985) FEBS Lett. 188,

Zalensky, A. O., Tuturova, K. F., Odintsova, N. A,, and Zalenskaya, I. A. (1982)

", "" ""

243-247

Stud. Biophys. 8 7 , 137-138