THE JOURNAL OF BIOLOCKAL CHEMISTRY 8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 12, Issue of April 25, pp. 6854-6659, 1990 Prmted in U.S.A.

Purification and Structure of Caltrin-like Proteins from Seminal Vesicle of the Guinea Pig*

(Received for publication, August 21, 1989)

Carlos E. Coronel$, Jovenal San Agustin, and Henry A. Lardyg From the Institute for Enzyme Research and Department of Biochemistry, The University of kiscon&, Madison, Wisconsk 53705

Two different small proteins that cross-react with the antiserum against bovine caltrin (calcium trans- port inhibitor) have been purified from the seminal vesicle contents of the guinea pig. The primary struc- ture and some molecular characteristics of the pure proteins are reported.

The two proteins interact with concanavalin A indi- cating the presence of carbohydrates in their mole- cules. Chemical deglycosylation with trifluorometh- anesulfonic acid, after reduction and carboxymethyl- ation, results in complete loss of affinity for the lectin. Removal of sugar components from the structure de- stroys the ability of caltrin-like proteins to react with antibodies to bovine caltrin.

The protein moving faster on polyacrylamide gel electrophoresis is designated guinea pig caltrin I, the other is II. They contain 45 and 55 amino acids, and the molecular weights of the peptide portions are 5082 and 6255, respectively.

Although they have entirely different amino acid sequences, they share some common features: recog- nition by rabbit antibodies to bovine caltrin, the pre- dominance of basic residues and the presence of 3 cysteine residues in fraction I and 8 in fraction II. The proteins have p1 values of 9.5 and 10.2, respectively, which are consistent with the amino acid composition.

The two pure fractions are approximately equally effective, on a weight basis, as inhibitors of 45Ca2+ uptake by guinea pig spermatozoa. The data presented reinforce the hypothesis that caltrin-like proteins are responsible for the previously reported (Coronel, C. E., San Agustin, J., and Lardy, H. A. (1988) Biol. Reprod. 38, 713-722), calcium-transport inhibitor activity de- tected in reproductive tract fluid from adult male guinea pigs.

Mammalian spermatozoa are unable to fertilize eggs at the time of mating and must spend some time in the female reproductive tract before they acquire that ability. During this period, sperm undergo biochemical, morphological, and phys- iological changes which, together, comprise a complex process termed “capacitation” (Chang, 1984; Austin, 1985). To be- come capacitated sperm must take up extracellular calcium

* This work was supported by Grant AM 10334 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Postdoctoral Fellow of the National Council for Scientific Re- search (CONICET), Argentina.

5 To whom correspondence should be addressed: 1710 University Avenue, Madison, WI 53705. USA Telephone (608) 262-3372.

to facilitate development of the “acrosome reaction.” The acrosome is an organelle covering the anterior portion of the sperm head, enclosed by its own membrane under the sperm plasma membrane and contains hyaluronidase, the proteolytic enzyme acrosin, and other hydrolytic enzymes. At the time of ovulation, mammalian ova are covered with a gelatinous protein layer, the zona pellucida, and surrounded by cell layers collectively known as the cumulus oophorus. During capaci- tation, the outer acrosomal membrane fuses with the overlying plasma membrane to form vesicles. When these membranes are completely discomposed (Barros et al., 1967) the stored hydrolytic enzymes are released and digest a path through the zona and cumulus materials permitting the sperm to penetrate the egg. In the guinea pig only acrosome-reacted spermatozoa can bind to and pass through the zona pellucida (Huang et al., 1981).

Accumulation of Ca2+ by ejaculated sperm is also necessary to promote “hyperactivation” of motility (Yanagimachi and Usui, 1974; Suarez et al., 1983,1984; Cooper, 1984). Ejaculated spermatozoa swim with moderate speed in a progressive linear direction. By this motion, sperm move from the vagina to the oviduct where they encounter the eggs. Calcium causes a change in the motility pattern and the new movement is characterized by a more rapid beat and wider excursion of the tail. The sperm describe a circular swimming trajectory that appears to aid penetration of the egg investments (Katz et al., 1978).

The development of these Ca’+-dependent events is sup- ported by the calcium in genital fluids. Calcium is present in millimolar concentrations in male as well as in female repro- ductive tract fluids (Olds and Van Denmark, 1957; Mann and Lutwak-Mann, 1981). Nevertheless, calcium movement across the plasma membrane appears to be extremely re- stricted in spermatozoa from different species. Epididymal spermatozoa from guinea pig, rabbit, and dog do not take up extracellular calcium spontaneously. In the guinea pig, cal- cium permeability can be induced by incubating the sperm for 20 min in a Ca’+-free medium containing lactate and/or pyruvate (Coronel and Lardy, 1987). The spermatozoa are then able to take up exogenous calcium and can develop the acrosome reaction within lo-15 min of exposure to Ca2+ (Singh et al., 1978; Hyne and Garbers, 1981). In contrast, metabolizable sugars that are present in the genital fluids prevent the enhancement of Ca2+ permeability and delay the acrosome reaction (Coronel and Lardy, 1987; Rogers and Yanagamachi, 1975). It has been proposed that these sugars play a specific role as “decapacitation factors” to control the development of the acrosome reaction until the spermatozoa encounter the eggs in the oviduct (Coronel and Lardy, 1987).

Control of Ca2+ transport into spermatozoa also involves the participation of specific proteins from the reproductive tract. A small (Mr 5411, Lewis et al., 1985), basic protein that

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inhibits calcium transport into epididymal spermatozoa has been isolated from bovine seminal plasma (Rufo et al., 1982). This calcium transport inhibitor, named caltrin, binds to the plasma membrane over the acrosome and principal tail re- gions of bovine spermatozoa but not to the post acrosomal area or the midpiece (San Agustin et al., 1987). It has been proposed that caltrin plays a key role timing the capacitation process in bovine spermatozoa (Lardy et al., 1988).

Caltrin-like proteins are present in reproductive tract fluid and seminal vesicle content of the guinea pig (Coronel et al., 1988). Both preparations blocked the enhancement of calcium permeability of the spermatozoa induced by incubation in a medium for capacitation and inhibited 45Ca2+ uptake by prein- cubated cells. The present paper describes a purification pro- cedure and structural characterization of two molecular forms of caltrin-like proteins from the guinea pig.

MATERIALS AND METHODS’

RESULTS

Cabin Purification-Two forms of caltrin-like proteins are present in the reproductive tract fluid and seminal vesicle content of the guinea pig (Coronel et al., 1988). Crude extracts lose all ability to inhibit Ca2+ uptake by spermatozoa when heated to 60 “C for 5 min, but dialyzed extracts are not affected by this heat treatment. We assume that caltrin-like proteins are destroyed by ion-dependent proteases present in the crude extract, which in the dialyzed samples have been rendered inactive (Coronel et al., 1988). Thus, dialysis and heating were introduced as routine steps.

The seminal vesicles of 10 adult guinea pigs (body weight -700 g) were removed and their content suspended in 110 mM NaCl, 2.5 mM benzamidine, 1 mM EGTA,’ and 25 mM

NaHC03 at pH 7.6 (extraction buffer). The suspension was centrifuged at 17,000 x g for 30 min at 2 “C and the super- natant (170 ml; 21.5 mg protein/ml) was dialyzed overnight at 4 “C against the same buffer. After removing denatured proteins by centrifugation at 27,000 X g for 30 min at 2 “C!, the supernatant was heated at 60 “C for 5 min and centrifuged again under the same conditions.

The supernatant (18 mg protein/ml) was brought to 30% saturation with solid ammonium sulfate, stirred for 30 min, and allowed to stand for 1 h at 0 “C. The protein pellet, collected at 23,700 X g for 30 min at 2 “C, was discarded and the supernatant was made 70% saturated with solid ammo- nium sulfate. The sample was left overnight at 4 “C and then centrifuged as above. The pellet was dissolved in a minimum volume of extraction buffer and dialyzed against 110 mM

NaCl and 25 mM NaHC03 at pH 8.0 (elution buffer). The sample was loaded onto a column (2.6 x 115 cm) of

Sephadex G-50 super fine (Pharmacia LKB Biotechnology Inc.) equilibrated and eluted with elution buffer. The fractions containing caltrin-like proteins, which were detected by im- munoblotting using a polyclonal anti-bovine-caltrin anti- serum, were pooled and brought to 90% saturation with solid ammonium sulfate. The protein pellet collected by centrifu- gation was resuspended in minimum volume of elution buffer and dialyzed overnight against the same solution.

The sample was submitted to a second cycle of gel filtration

’ The “Materials and Methods” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverlv Press.

on Sephadex G-50 super fine using the same column and the same elution conditions as in the first step. Four distinct protein peaks were apparent with caltrin-like proteins in the major peaks 3 and 4. The fractions containing caltrin-like proteins were pooled and made 90% saturated with solid ammonium sulfate and treated as in the first cycle.

The sample was subjected to a third step of gel filtration on Sephadex G-50 super fine using a column (1.2 x 107 cm) equilibrated and eluted with the bicarbonate buffer. Two overlapping protein peaks containing caltrin-like proteins were obtained (Fig. l), and designated fractions I and II. Fractions 38-49 were pooled and concentrated by ultrafiltra- tion in an Amicon pressurized chamber through a Diaflo YM- 2 membrane or by precipitation with solid ammonium sulfate to 90% saturation.

At this stage of purification the isoelectric points of the caltrin-like proteins of fractions I and II were determined to be basic and minor contaminants were acidic. This permitted a purification step involving cation exchange chromatogra- phy. The concentrated samples were dialyzed against 25 mM

Tris-HCl, pH 7.6, to remove NaCl or ammonium sulfate depending on the procedure used to concentrate the proteins. The samples were loaded onto a column (0.5 X 15 cm) of carboxymethyl-cellulose (CM-32, Whatman, United King- dom), equilibrated with 25 mM Tris-HCl, pH 7.6. The inhib- itor proteins were retained in the column while those with lower p1 values were removed with starting buffer (Fig. 2, peaks A and B). These protein fractions moved as caltrin-like proteins when submitted to SDS-PAGE (Fig. 3, lanes A and B). To elute the proteins bound to the resin, a stepwise NaCl gradient from 0.1 to 0.4 M in equilibration buffer was applied. Caltrin-like protein fraction I was eluted with 0.1 M NaCl (Fig. 2, peak D; Fig. 3, lane D). In trace amounts two different proteins with intermediate electrophoretic mobility between fraction I and II of caltrin-like proteins were eluted with 0.2 M NaCl (Fig. 2,peaks E and F; Fig. 3, lanes E and F). Caltrin- like protein fraction II was removed from the column with 0.4 M NaCl (Fig. 2, peak G; Fig. 3, lane G).

Antigenic Actiuity-Antigenic activity of all eluted protein peaks was assessed by Western blotting. Anti-bovine caltrin antiserum reacted with three different proteins, fractions I and II (Fig. 4, lanes D and G) and a third protein fraction, peak E, (Fig. 4, lane E). The latter protein moves behind of, but very close to, fraction I (Figs. 3 and 4). Because of this, it was impossible to identify that third component by SDS- PAGE and Western blotting, without previous chromato-

2ti

o 1 2 a

’ The abbreviations used are: EGTA, [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; IEF, isoelectric focusing; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

fractions FIG. 1. Elution profile from Senhadex G-50 column (third

step). A column (1.i X 107 cm) was equilibrated with 110 mM ‘NaCl, 25 mM NaHCO? at pH 8.0. Samples eluted between fractions 38-48 were pooled and concentrated.

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6856 Purification and Structure of Caltrin-like Proteins

3tio0.5

FIG. 2. Elution profile from CM-cellulose column. Pooled Sephadex G-50 fractions containing the two molecular forms of caltrin-like proteins were loaded onto a CM-cellulose column equili- brated with 25 mM Tris-HCl at pH 7.6 and eluted as described under “Materials and Methods.” Peaks n and C; represent fractions 1 and II, respectively. Peak E is also immunoreactive with anti-bovine caltrin antiserum.

¶m- w--- ABCDEFGH

FK. 3. SDS-PAGE of protein peaks eluted from CM-cellu- lose column. The electrophoresis was performed in a 15% acrylamide gel and the proteins were stained with Coomassie Brilliant Blue R- 250. Each lane contains a sample from the corresponding eluted peak of Fig. 2. Lane D, fraction I; lane G, fraction II; lane H, molecular weight marker proteins (Bio-Rad): phosphorylase b (92,500); bovine serum albumin (66,200); ovalbumin (45,000); carbonic anhydrase (31,000); soybean trypsin inhibitor (21,500), and lysozyme (14,400).

graphic separation. The purified proteins were desalted by gel filtration on Sephadex G-50 super fine in a column (1 X 26 cm), equilibrated with 12.5 mM NH.,HCOa, freeze-dried, and stored at -60 “C.

Purity of Proteins-Purity of proteins in peaks D, E, and G, was checked using a gradient HPLC system. All three proteins appeared homogeneous when submitted to reverse- phase HPLC as indicated under “Materials and Methods.” These desalted samples were used for sequencing.

Isoelectric Point Determination-The migration of partially purified fractions I and II in acrylamide isoelectric focusing gels was compared with several proteins of known isoelectric points. The isoelectric points calculated for caltrin-like pro- teins are 9.5 for fraction I and 10.2 for fraction II. Similar results were obtained with an agarose gel isoelectric focusing system.

We previously reported acidic p1 values (5.3, 6.0, and 6.2) for three fractions of caltrin-like proteins in the reproductive tract fluid of the guinea pig (Coronel et al., 1988). The exper- iments were carried out by focusing the crude samples on

A BCD EFGH

FIG. 4. Western blotting of protein peaks eluted from CM- cellulose column. SDS-PAGE was performed in 15% acrylamide gels, and the proteins were then transferred to nitrocellulose mem- branes by electrophoretic transference. Caltrin-like proteins were reacted with anti-bovine-caltrin antiserum, as indicated under “Ma- terials and Methods,” and detected with anti-rabbit IgG conjugated with alkaline phosphatase. Peaks D, E, and G (lanes D, E, and G), were recognized by the antiserum. Lanes D and G correspond to fractions I and II, respectively. Lane H corresponds to prestained marker proteins (Bio-Rad).

agarose gels. Those values do not agree with data of the present paper calculated using the purified proteins. To find an explanation for these different results, the protein pattern of the reproductive tract fluid and seminal vesicle content was analyzed in an acrylamide gel isoelectric focusing system and compared with those of partially purified fractions. Proteins with electrophoretic mobility corresponding to those of cal- trin-like proteins were not detected either in reproductive tract fluid or in seminal vesicle content. Both crude sources contain several protein fractions with similar p1 values to those reported in the previous paper (Coronel et al., 1988). It is assumed that, in the reproductive tract fluid, caltrin-like proteins interact with more acidic components which mask their p1 values.

In the case of seminal vesicle contents, caltrin-like proteins seem to be aggregated in a more complex structure. Precipitin complex formation, in Ouchterlony double immunodiffusion tests, were observed only when the samples had been treated with at least 1% SDS. No immunoreaction was detected at lower SDS concentration.

Primary Structure-The amino acid sequences of fractions I and II were determined on unaltered samples. Forty-five residues were detected in fraction I. Seven were basic amino acids and four acidic. In fraction II, which is formed by 55 residues, 11 were basic while only 3 were acidic amino acids. Although more than 80% of total amino acids were easily identified in both molecules, 5 residues in fraction I and 10 amino acids in fraction II could not be identified which indicated the presence of carbohydrates bound to the peptidic chain, and/or -S-S- bridges between cysteine residues.

To investigate the presence of carbohydrates in the struc- ture of these proteins, their ability to interact with concana- valin A was assessed. Concanavalin A labeled with biotin, and avidin conjugated with alkaline phosphatase or horseradish peroxidase, were used for blotting on nitrocellulose mem- branes. Both fraction I and II of caltrin-like proteins bound the lectin indicating the presence of carbohydrates in their molecules (data not shown). This contrasts with bovine cal- trin which contains no carbohydrate (Rufo et al., 1982). To remove the carbohydrate moiety of caltrin-like proteins of the guinea pig, chemical deglycosylation with trifluoromethane-

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Purification and Structure of Caltrin-like Proteins 6857

sulfonic acid was applied (Sojar and Bahl, 1987). This tech- nique effectively deglycosylates ovalbumin but caltrin-like proteins treated with trifluoromethanesulfonic acid main- tained their ability to interact with concanavalin A, indicating that carbohydrates were still present in the molecules. Com- plete deglycosylation of caltrin-like proteins was successful only when the proteins were previously submitted to reduction and carboxymethylation. Both fractions were alkylated with iodoacetic acid after reduction with dithiothreitol (Allen, 1981).

The chemical modifications permitted the identification of 3 cysteine residues in positions 14, 24, and 32 in fraction I. In fraction II, 8 cysteines located in positions 14, 21, 30, 36, 42, 43,47, and 51 were recognized (Fig. 5).

CNBr cleavage was applied to fraction II to facilitate the sequencing analysis of the carboxyl-terminal portion. After treatment with CNBr, two sequences were observed: one from the NH2 terminus and another following the methionine at residue 18. This pattern was consistent with the presence of only one methionine in the peptide. The CNBr strategy identified 37 additional residues after the first 18.

The amino acid at the NH, terminus of fraction II could not be identified in the unmodified protein. After CNBr treatment this NH*-terminal amino acid was identified as arginine. One possible explanation for this unexpected result is that CNBr treatment removed some modification of the arginine side chain that caused a blank cycle in the original sequence. The modification is not to the terminal amino nitrogen as that would have prevented sequencing. It is also possible that sugars can be bound to the polypeptide on that residue.

According to the primary structure of both molecules there are two sites where sugars could be linked to the polypeptides. In the case of fraction I, these sites would be located among the residues -13-14-15- (-Asn-Cys-Ser-) and -41-42-43- (-Asn- Arg-Ser-). In fraction II, sugars could be bound to the guani- dino group of arginine at the NH2 terminus and also in the portion formed by residues -28-29-30-31- (-Asn-Lys-Cys- Thr-) (Fig. 5).

Bovine Caltrin 10

To verify that the end of the two peptides had been reached in the sequencing procedure, carboxyl-terminal residues were determined using carboxypeptidase Y according to Hayashi (1977). The yield of products at different periods of time confirmed the sequences shown in Fig. 5.

The amino acid sequence of a third protein eluted from the CM-cellulose column and recognized by anti-bovine caltrin antiserum (peak E, Figs. 2 and 3), was determined. The protein contains 51 residues, 8 basic and 3 acidic. The scheme presented in Fig. 5 shows that the sequence of identified amino acids in fraction E is the same as that of fraction II from residue 5 to the COOH terminus. Fraction E was used in sequencing studies without chemical modifications. Under these conditions 8 residues could not be identified, but they correspond with the 8 cysteine residues located at sites 14,21, 30, 36, 42, 43, 47, and 51 of fraction II (Fig. 5). It is therefore highly likely that fraction E represents a molecular form of fraction II which has lost four amino acids from the NH2 terminus by proteolysis.

Reduction, carboxymethylation, and deglycosylation of the caltrin-like proteins affected the electrophoretic mobility and the antigenic activity. When these modified proteins were subjected to SDS-PAGE and Western blotting, fraction II ran in a tight band slower than the native form with electropho- retie mobility corresponding to a protein of 12,000 Da (data not shown). No alteration in the mobility of fraction I was detected. However, the two proteins lost the antigenic activity with anti-bovine-caltrin antiserum.

Inhibition of Calcium Uptake-To study the effect of pure caltrin-like proteins on the uptake of extracellular calcium by guinea pig spermatozoa, the permeability of the cells to Ca*+ must be induced by a preliminary incubation in a Ca*+-free medium (Coronel and Lardy, 1987). For this purpose, epidi- dymal spermatozoa were incubated in minimal capacitation medium-pyruvate/lactate at 37 “C for 90 min, and their ability to take up 45Ca2’ was then assessed in the presence of different amounts of fractions I and II of caltrin-like proteins. At low protein concentrations, 0.05 and 0.1 mg, fraction I was a

20 30 Scr.Asp.Glu.Lys.Ala.Scr.l’ro.Asp.Lys.Hir.His.Arg.l’he.Scr.Lcu.Ser.Arg.Tyr.Ala.Lys.Lcu.Ala.Asn.Arg.Leu.Ala.Asn.Pro.Lys.Lcu.Leu.Glu.Thr.Phc.Leu

40 .Ser.Lys.Trp.Ilc.GIy.Asp.Arg.Gly.A~n.Arg.Scr.V~l

Guinea Pig Caltrin I 10 20 30

Ala.Phe.Ala.Pro.Ser.Lys.Val.Asp.Ser.Asp.Arg.Pro,Asn.Cys.Ser.Arg.Tyr.Val.Gln.His.Leu.Tyr.Met.Cys.Thr.Lys.Glu.Lcu.Asp.Pro.Val.Cys,Gly.Th~,A~p, 40

.Gly.His.Thr.Tyr.C.Iy.Asn.Arg.Scr.Ile.Phe

Guinea Pig Caltrin II 10 20 30

Arg.Arg.Leu.His.Gly.Gln.Ala.Ile.Asn.Arg.Pro.Gly.Ser.Cys.Pro.Arg.Val.M~t.~lc.Ty~.Cy~.P~o.Al~.Arg.Hi~.Pro.Pr~.Asn.Lys.Cys.Thr.Ser.Asp.Ty~.A~p. 40 50

.Cys.Pro.Lys.Pro.Gln.Lys.Cys.Cys.Pro.Gly.Tyr.Cys.Gly.Lys.Gln.Cys.Tyr.Gln.Pr~.Glu

“Peak F” 1 I 6 lb 26

.__............,.._........ Gly.Gln.Ala.lle.Asn.Arg.Pro,Gly.Scr.Xxx.l’fo.Arg.Val.Met.lle.Tyr.Xxx.Pro.Al~.Arg.His.Pro.Pr~.Asn.Lys.Xxx,Thr,Sc~.A~p.Ty~.A~p. 36 46

.Xxx.Pro.Lys.Pro.Gln.Lys.Xxx.Xxx.Pro.Gly.Tyr.Xxx.Gly.Lys.GIn.Xxx.Tyr.Gln.Pro.Glu

FIG. 5. Sequence of bovine caltrin and caltrin-like proteins from the guinea pig. The sequence of bovine caltrin is that reported by Lewis et al. (1985).

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Purification and Structure of Caltrin-like Proteins

00 01 0.2 0.3 04 05 0.6

mg of protein

FIG. 6. Effect of pure caltrin-like proteins upon %a’+ up- take into preincubated epididymal guinea pig spermatozoa. The cells were incubated in Ca’+-free medium for capacitation for 90 min and then manipulated as detailed under “Materials and Meth- ods.” The spermatozoa were resuspended in fresh medium containing different amounts (O-O.5 mg/lOR cells) of caltrin-like protein fraction I (@), or fraction II (O), and then incubated for 10 min in presence of 1 mM CaCl* labeled with ‘%a”. The cells were separated and the accumulated radioactivity was counted. “Induced Ca-uptake” was calculated by subtracting the value obtained with fresh cells from that obtained with preincubated spermatozoa. Data shown are the mean of triplicate determinations.

somewhat more effective inhibitor of calcium uptake than fraction II (Fig. 6). At greater amounts of protein, 0.2 and 0.5 mg, fraction II was slightly more effective. Sperm calcium uptake was inhibited about 40% by 0.5 mg of fraction I/10’ sperm while the same amount of fraction II produced 48% inhibition.

DISCUSSION

Inhibitory activity on sperm calcium uptake has been de- tected in the reproductive tract fluid and seminal vesicle content of the guinea pig (Coronel et al., 1988). Those fluids contain two small proteins similar to one isolated from bovine seminal plasma named “caltrin” (Lewis et al., 1985). Anti- bovine-caltrin antibodies cross-react with the two proteins from the guinea pig. Because of this, they have been desig- nated “caltrin-like proteins.”

The present paper details a purification procedure for cal- trin-like proteins from seminal vesicle contents of the guinea pig. Some structural and biochemical characteristics of the pure fractions have been determined. In addition to the main proteins I and II, a third component (designated E) recognized by anti-bovine caltrin antibodies was separated by gradient elution from carboxymethyl cellulose. It represents a degra- dation product of II from which four amino acids have been removed from the NH, terminus. A minor contaminant in fraction I (D in Fig. 3) is identical with I except that a single amino acid is missing from the NH2 terminus. Both of these are assumed to be proteolytic degradation products of the parent proteins II and I, respectively. These proteins run very close to one another in SDS-PAGE, but two bands can be visualized using 15% acrylamide as separating gel.

In a previous paper, where these two calcium transport inhibitory proteins were first observed, we suggested the pos- sibility that the smaller protein (I) might have been derived from II with retention of its ability to inhibit calcium trans- port. Determining the sequence of the two proteins establishes that they are not structurally related.

Bovine caltrin is also not structurally related to either of the guinea pig caltrin-like proteins except for a sequence Gly- Asn-Arg-Ser near the COOH terminus in both bovine caltrin

and guinea pig caltrin I. The ability of rabbit anti-bovine caltrin antibodies to react with each of the guinea pig proteins must depend on the formation of similar shape and charge groups in the tertiary structure of the caltrins. The loss of recognition by the antibodies when the guinea pig caltrins have been reduced, carboxymethylated, and deglycosylated indicates a role for disulfide bonds and/or carbohydrate resi- dues in establishing conformations recognized by antibodies induced by a protein devoid of both carbohydrate and sulfur- containing amino acids.

Despite the lack of structural similarity between bovine caltrin and the two guinea pig caltrin-like proteins they pos- sess some common features. They are probably synthesized in the seminal vesicles for they are found only there and in seminal fluid. All three are small basic proteins with compa- rable molecular weights. Caltrin and the analogs from guinea pig do not bind calcium but affect its movement across sper- matozoal membranes. Because of these similarities we propose to name the two newly described proteins guinea pig caltrin I and II.

Bovine caltrin binds to bull spermatozoa over the acrosome and to the tail (San Agustin et al., 1987). Thus, caltrin may function by preventing a premature development of the ac- rosome reaction by blocking Ca2+ influx into the cells. On the tail, caltrin may suppress Ca*+-dependent hyperactivation of motility by inhibiting Ca2+ penetration until the sperm reach the oviduct (Lardy et al., 1988). In this part of the genital tract, the ionic environment may lead to conformational changes in the molecule as a result of specific ligand loss (San Agustin and Lardy, 1990). Bovine caltrin loses its inhibitory activity when separated from anions that accompany it in seminal fluid, it then becomes an enhancer of calcium trans- port (San Agustin and Lardy, 1990). Enhancer activity has not been observed with guinea pig caltrins. Some other means of relieving the inhibition of calcium uptake must have evolved for the guinea pig in order to permit the uptake of calcium required to induce the acrosome reaction.

Relief of the inhibition of calcium uptake by the sperm tail may lead to the hyperactive motility that is also necessary for sperm penetration of the layers surrounding the egg (Yana- gimachi and Usui, 1974). It will be interesting to determine how both caltrins I and II bind to guinea pig spermatozoa, i.e. whether one is adapted to bind over the acrosome and the other to the tail or whether each is capable of binding at both sites.

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C E Coronel, J San Agustin and H A Lardyguinea pig.

Purification and structure of caltrin-like proteins from seminal vesicle of the

1990, 265:6854-6859.J. Biol. Chem. 

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