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Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems
Journal o[ Reproductive Immunology, 11 (1987) 307-319
307
Elsevier Scientific Publishers Ireland Ltd.
JRI 00490
Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems Mark S. Siegel, Dana S. Bechtold, Janet L. Willand and Kenneth L. Polakoski Department of Obstetrics and Gynecology, Washington Universilty, St. Louis, Missouri (U.S.A.) (Accepted for publication 29 May 1987)
Summary Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55 000), alpha-acrosin (45 000-49 000) and beta-acrosin (34 000-38 000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological ditterences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar
Correspondence to: Kenneth L. Polakoski, Department of Obstetrics and Gynecology, Washington University, 4911 Barnes Hospital Plaza, St. Louis, MO 63110, U.S.A. 0165-0378/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
308
proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34 000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm. Key words: proacrosin, acrosin, proteinase, immunology, spermatozoa.
Introduction
Acrosin (EC 3.4.21.10) is a sperm proteinase with trypsin-like specificity that is believed to have a critical role in events leading to fertilization. Proacrosin is a zymogen precursor to acrosin and is the form that accounts for the majority of the acrosin that is present in epididymal and freshly ejaculated sperm. From studies on proteins isolated from boar sperm, a model system has been devised which assists in the design of investigations concerning the numerous components of the proacrosinacrosin system (reviewed by Polakoski and Siegel, 1986). The porcine species has been utilized for many of these studies because large amounts of material are available and the individual components are obtained in relatively stable forms. We have recently purified proacrosin from human sperm (Siegel et al., 1986). The partial biochemical characterization of this key protein has resulted in the observation of a number of similarities between the human and boar proacrosin-acrosin systems. These include similar molecular weights and interrelationships between the different forms of proacrosin and acrosin. Even though considerable progress has been made regarding the biochemical characterization of the proacrosin-acrosin system, little immunological information on any of its components is available. Furthermore, there are presently no reports on antibodies developed specifically to proacrosin. This is particularly surprising since immunochemical approaches have proven increasingly useful in probing the structural features of proteins (Kilgore et al., 1986) which may be particularly important in the regulation of this critical enzyme system. In the present investigation, polyclonal antibodies were used to provide a generalized view of the immunochemical identities of human and boar proacrosin. The data obtained are of particular importance because they
309
clearly demonstrate that there are immunochemicai differences between these species. These differences may be of particular interest in investigating the regulatory components of this system as well as in the design of immunochemical approaches to contraception. Materials and Methods
Reagents N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), benzamidine, dithioerythritol, dithiothreitol (DTT), Freund's incomplete adjuvant, 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA) and iodoacetamide were obtained from Sigma, St. Louis, MO. Glycine, Tris(hydroxymethyl)aminomethane (Tris), polyoxethylene sorbitol (Tween 20), acrylamide, 4-chloro-1-naphthol (HRP color reagent) and goat anti-rabbit horseradish peroxida~e conjugate were purchased from Biorad, Richmond, CA. Sodium dodecyl sulfate (SDS) and guanidine-HC1 were obtained from Pierce, Rockford, IL and ultrapure ammonium sulfate from Schwarz-Mann Inc, Spring Valley, NY.
Preparation and purification of human and boar proacrosin Human sperm were obtained from freshly ejaculated semen samples. The sperm were washed, acid extracted and proacrosin was purified as previously described (Siegel et al., 1986). Boar sperm were obtained by flushing the sperm through the caudae epididymidis with Hepes buffer (0.1 M) which contained benzamidine (0.05 M) at pH 7.5. The sperm were then washed free of epididymal fluid by centrifugation through 0.05 M benzamidine in 1.0 M sucrose (pH 6.0). The washed sperm were acid extracted and the proacrosin was partially purified via a pH 6.0 and then a 35% ammonium sulfate precipitation according to the procedures of Polakoski and Parrish (1977) as modified by Kennedy et al. (1981). The ammonium sulfate precipitated material was then further purified via a Superose 12 gel filtration procedure using a Pharmacia FPLC system as described for human proacrosin (Siegel et al., 1986). The peak fractions of proacrosin were dialyzed, pooled and used for the activation and immunoblotting experiments that are described below. However, to prevent proacrosin autoconversion from occurring during the antibody development, the samples that were used for raising the antibodies were treated in a slightly different manner prior to the Superose-12 gel filtration. For these studies each of the human and boar preparations were first reduced and then alkylated by a modification of the procedure described by Lane (1978). Briefly, the samples wcrc adjusted to a final concentration of 0.3 mg/ml in 6 M guanidinc-HCI at pH
310
2.5. The proteins were then reduced by the addition of 1/50 vol. of fresh 0.25 M dithioerythritol, heated for 2 min at 100°C and then cooled to 22°C. One twentieth volume of 0.25 M iodoacetamide was then added to the sample and incubated at 50°C for 15 min. The sample was then dialyzed against 1000-fold excess of double distilled water and iyophilized. The material was then resuspended in 200 ~1 of 8 M guanidine in 0.5 M acetic acid (pH 2.5) and injected onto the column.
Electrophoresis of proacrosin and acrosin Two micrograms of the purified human and boar proacrosin were autoactivated for 0, 5, 15, and 60 min by adjusting the pH to 8.0 with Tris buffer (final concentration 0.05 M). The autoactivation was stopped by the addition of an equal volume of sample buffer containing 4% SDS, 20% glycerol, 0.5 M DTT and the solutions were boiled for 2 min. Duplicate aliquots of each of these preparations were electrophoresed on two adjacent 12.5% discontinuous polyacrylamide gels (Laemmli, 1970) using a Hoefer Electrophoresis apparatus. Following electrophoresis for 3.5 h at 20 mA/gel, one of the gels was stained for protein using a silver staining procedure (Wray et al., 1981) so that the molecular weights of the proacrosin and acrosin could be determined. Proteins from the duplicate gel were transblotted and the resulting blots treated as described in the western blotting section below.
Production of human and boar proacrosin antibody Female New Zealand white rabbits were injected intradermally on day 0 and at two more times at 2-week intervals with either the purified human or boar proacrosin which was reduced and alkylated as described above. The proacrosin preparations (50 ~g) were mixed with equal volumes of Freund's incomplete adjuvant prior to injection. On the 9th day following the last injection, 50 ml of whole blood was collected from the rabbit ear vein, allowed to coagulate at room temperature for 4 h and the blood sera was then obtained by centrifugation of the blood at 10 000 x g for 10 min. The antibodies were obtained from immunized and pre-immunized sera and partially purified by a 40% ammonium sulfate precipitation for 1 h at 4°C. The samples were centrifuged and the resulting pellet was resuspended in 0.01 M Tris at pH 7.5 and subsequently dialyzed against the same buffer to remove the salts.
Western blotting Immunoblotting was performed with a slight modification of the procedure of Towbin et al. (1979). For these experiments, the proteins were electrotransferred at 4°C in a 0.03 M MES, 0.19 M glycine buffer (pH
311
6.1) for 16 h at 30 V and then for 2 h at 50 V. The sheets were incubated for 2 h with the primary antibody preparation diluted either 1 : 25 (antihuman proacrosin) or 1 : 100 (anti-boar proacrosin) in the washing buffer containing 1% BSA. Preliminary experiments showed that these were the optimal concentrations for each of the primary antibodies. Results
The SDS-PAGE analysis of proacrosin and its activation products have been individually described for both the human (Siegel et al., 1986) and boar (Polakoski and Parrish, 1977). The results shown in Figs. 1 and 2 are
~,i,i~,~~,i~,i~,~i~,i,i,~,i,~,i i,,,i¸i~i,i~,i,i~!,i~,!~i~ IIIIIIIIII--BSA
PRO-ALPHA-
--A LD BETA---CA
-CYT A
B
C
C
D
Fig. 1. SDS-polyacrylamide gel electrophoretic analysis of the time course of human proacrosin autoconversion to acrosin. Equal volumes containing 2 Ixg of purified proacrosin and 0.1 M Tris (pH 8.0) were incubated for 0rain (lane A), 5 rain (lane B), 15 rain (lane C) and 60min (lane D). The reaction was stopped by the addition of 2% SDS/0.25 M dithiothreitoi and incubated for 2 min at 100°C. The samples were subjected to SDS-PAGE and the gels were silver stained for protein as described in Materials and Methods. The proacrosin (PRO), alpha-acrosin (ALPHA) and beta-acrosin (BETA) migrated as indicated. The standard proteins were (BSA, Mr, = 68 000), aldolase (ALD, M, = 40 000), carbonic anhydrase (CA, M, = 29 000) and cytochrome c (CYT C, M, = 12 800).
312
PRO-~ ALPHA-
-BSA i
BETA-
m ==
-ALD m
-CA
-CYT A
B
C
C
D
Fig. 2. SDS-polyacrylamide gel electrophoretic analysis of the time course of boar proacrosin autoconversion to acrosin. Purified boar proacrosin was autoactivated for 0 min (lane A), 5 min (lane B), 15 rain (lane C) and 60 min (lane D). Two micrograms of protein per lane were electrophoresed and the gels processed as described in the legend to Fig. 1.
presented in order to directly compare the major components of the proacrosin-acrosin system between these species and to demonstrate the relative amounts of each form of acrosin that is analysed via immunoblotting in Figs. 3 and 4. Proacrosin autoconversion The results presented in Fig. 1, lane A, show that human proacrosin has an apparent molecular weight between 51 000 and 55 000. When this preparation is autoactivated under the conditions described, most of the proacrosin is converted to alpha-acrosin (Mr --49 000) within 5 min (lane B). During the subsequent incubation, beta-acrosin is detected and becomes the predominant form at 60 min.
313
H B
H
B
H
B
H
B
-BSA -ALD -CA
-CYT C
A
B
C
D
E
F
G
H
Fig. 3. lmmunoreaction of antibodies to human proacrosin towards the purified human and boar proacrosin-acrosin systems as determined by immunoblotting. Purified human (H) and boar (B) proacrosin were autoactivated for 0 rain (lane A and B), 5 min (lane C and D), 15 min (lane E and F) and 60 min (lane G and H) as described in the legend to Fig. 1. The samples (2 i~g/lane) were resolved by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose and probed with antibodies to human proacrosin as described under Materials and Methods.
The purified boar proacrosin activation profile (Fig. 2) was very similar to that described for human proacrosin. Although not easily observed, two bands of protein (M, = 53 000 and 55 000) were present in the highly purified boar proacrosin preparation (Fig. 2, lane A). Following proacrosin autoconversion for 5 min, apparently all of the proacrosin is converted to alpha-acrosin (M, = 45 000) (Fig. 2, lane B). After 60 min, most of the alpha-acrosin was converted to beta-acrosin (Fig. 2, lane D, .Mr,= 38 000).
314
H
B
H
B
H
B
H
B
--9SA
m
lid
--ALD
-CA
--CYT C
A
B
C
D
E
F
G
H
Fig. 4. Immunoblot of human and boar proacrosin and acrosin with antibodies to boar proacrosin. Human (H) and boar (B) proacrosin were autoconverted for various time intervals and analyzed for their immunoreactivity towards antibodies to boar proacrosin as described in the legend to Fig. 3.
Immunobloning An immunobiotting technique was utilized to analyse the specificity and sensitivity of rabbit antibodies directed to highly purified human and boar proacrosin. To determine the extent to which non-specific binding affects the immunoblotting procedure, control experiments were performed (in the absence of the primary antibody) with ammonium sulfate precipitated antibody from pre-immunized rabbit sera. In each of these cases there was no observed color reaction on the nitrocellulose sheets (results not shown) indicating that the reaction observed was not due to either a non-specific antibody interaction or to non-specific binding of the second antibody. The antisera to the human proacrosin was tested for its immunoreactivity with human proacrosin and acrosin as well as with the boar proacrosin and acrosin (Fig. 3). The human proacrosin antibody reacted with human proacrosin (lane A) as well as human alpha-acrosin (lanes B,
315
E and G). However, there was apparently no reactivity with the human beta-acrosin (lanes E and G). In addition, boar proacrosin (lane B) and to a lesser extent alpha-acrosin (lanes D and F) cross-reacted with the human proacrosin antibody (1 : 25 dilution), while the beta-acrosin did not (lanes D and F). The antibody to boar proacrosin (1 : 100 dilution) was also tested for its immunoreactivity with the human and boar proacrosin and acrosin (Fig. 4). There was no cross-reactivity observed between the human proacrosin, alpha-acrosin or beta-acrosin and the boar proacrosin antibody (lanes A, C, E and G) even at antibody dilutions of either 1:25 or 1:10 (results not shown). However, there was a strong immunoreaction between the boar proacrosin antibody and boar proacrosin (lane B) and
H
B
--ALD
-CA ii~ii~!~ ~,~i~~,~,~i~i~~
~ii~i~i i~!~~iI~i ~, ~ ~ ~
--CYT C A
B
316
H
B
H
B
-BSA -ALD -CA
-CYT
A
B
C
C
D
Fig. 5. (a) SDS-polyacrylamide gel electrophoretic analysis of non-purified acid extracts of human and boar sperm. Approximately 2 I~g of protein from non-purified human (lane A) and boar (lane B) sperm extracts were individually subjected to SDS-polyacrylamide get electrophoresis and the gels were silver stained for protein as described in Materials and Methods. (b) Immunoblot of unpurified human (H) and boar (B) sperm extracts using antibodies to highly purified human and boar proacrosin. The results of the immunoreaction of antibodies to human proacrosin against the sperm extracts (2 I~g/iane) are shown in lanes A and B. The immunoreaction of antibodies to boar proacrosin against the sperm extracts (2 p,g/lane) are shown in lanes C and D.
boar alpha-acrosin (lane D and F). A distinct but weaker reaction was observed for the boar beta-acrosin (lane H). Non-purified human and boar sperm extracts were used to determine if the antibodies reacted with other sperm antigens. In these preparations at least 95% of the total acrosin was present in the proacrosin form (results not shown). The human sperm extracts (Fig. 5a, lane A) were reacted with antibody to human proacrosin. The results shown in Fig. 5b (lane A)
317
demonstrate that the antibodies apparently reacted with 53 000, 49 000 and 34000 molecular weight antigens. Interestingly, several immunoreactive bands were detected at molecular weights above 68 000. However, when the boar sperm extracts (Fig. 5a, lane B) were reacted with the antibody to human proacrosin there were only two weakly immunoreactive bands (Mr = 55 000 and 53 000) observed. The boar proacrosin antibodies were also incubated with the nonpurified human and boar sperm extracts which had been electroblotted to nitrocellulose sheets. There was no cross-reactivity observed with the human sperm extract (Fig. 5b, lane C) while intense bands (Mr = 55 000 and 53 000) were observed in the boar extracts (Fig. 5b, lane D). Discussion
This report documents that: (a) in the human and boar proacrosinacrosin system there are similarities in the molecular weights of the main components as well as similar time courses for proacrosin auto-conversion; (b) antibodies can be produced against highly purified human and boar proacrosin; (c) human proacrosin antibodies that were developed cross-react with human proacrosin and alpha-acrosin while the boar proacrosin antibodies cross-react with boar proacrosin, alpha- and betaacrosin; (d) human proacrosin antibodies cross-react with boar proacrosin while the boar proacrosin antibodies do not react with the human proacrosin-acrosin system; (e) the antibodies that were developed were also shown to be highly specific for the proacrosin-acrosin system within each of the non-purified sperm extracts. As observed in Figs. 1 and 2, the molecular weights of the human and boar proacrosin-acrosin system are quite similar. The relative molecular weight for proacrosin is in the 51 000-55 000 range while alpha-acrosin is between 45 000 and 49 000 and beta-acrosin is about 34 000-38 000. In addition, the time sequence of autoconversion of proacrosin to acrosin was very similar in both species. Under the conditions employed, proacrosin conversion to alpha-acrosin occurred in less than 5 min while beta-acrosin was the predominant molecular weight form after 60 min incubation. Similar molecular weights and autoconversion times have been previously reported for the proacrosin-acrosin system from human (Siegel et al., 1986) and boar (Polakoski et al., 1977) sperm. However, the demonstration of the relative concentration of proacrosin and each of the forms of active acrosin at the different time points was required to estimate the cross-reactivity of the proacrosin antibodies in the western blotting experiments in Figs. 3 and 4. Although antibodies to acrosin from a variety of species have been
318
developed (reviewed by Polakoski and Siegel, 1986), no reports of proacrosin antibodies are available. The difficulty in making antibodies to proacrosin involved the probable autoconversion of proacrosin to active acrosin forms during the immunization process. This problem was alleviated by first reducing and then alkylating the proacrosin prior to the immunization thereby preventing proacrosin autoconversion. The ability of the proacrosin antibodies to cross-react with the specific components of the proacrosin-acrosin system was evaluated in Figs. 3, 4 and 5a. Interestingly, the antibodies to human proacrosin react with human proacrosin and alpha-acrosin but not with beta-acrosin (Fig. 3). This indicates that the antibodies that were raised against human proacrosin are directed to antigens or conformations that are lost during the final proteolytic conversion process which occurs during the formation of beta-acrosin from alpha-acrosin. This does not appear to be the case for the boar proacrosin antibodies since at least some of these react with the boar beta-acrosin (Fig. 4). It is not presently known if this is the result of significant differences in the structure of proacrosin from these two species. Notable differences exist with respect to the immune response of the human proacrosin antibodies with boar proacrosin and acrosin as compared to the boar proacrosin antibodies reactivity with human proacrosin and acrosin. The human proacrosin antibodies reacted predominantly with boar proacrosin, very slightly with the alpha-acrosin and not with beta-acrosin. The boar proacrosin antibodies, however, do not cross-react with any of the major components of the human proacrosin-acrosin system even at antibody concentrations ten-fold higher than these used in Fig. 4. It is possible that there are specific antigenic sites on human proacrosin-acrosin which are not common to the boar proacrosin-acrosin system. This, therefore, would permit an immunoreaction with human proacrosin antibody and human proacrosin but not the boar proacrosin antibody with human proacrosin. We are presently attempting to purify sufficient material from each of these proacrosin-acrosin systems to sequence these proteins so that it may be determined if the primary structural differences in these molecules could account for the observed immunological differences. The specificity of antibodies produced to the proacrosin-acrosin system are shown in Fig. 5a and b. The vast molecular weight range of proteins present in human and boar sperm extracts is shown in Fig. 5a. Despite the large numbers of proteins present, the major antigens that react with the antibodies to human proacrosin have relative molecular weights of 53 000-55 000 (Fig. 5b). The results shown in Figs. 1 and 3 strongly suggest that these antigens are proacrosin. However, minor
319
bands of reactivity were also observed in regions that correspond to molecular weights of 68 000 and above as well as at 49 000 and 34 000. It is not presently known if these molecular weight forms are part of the human proacrosin-acrosin system or represent possible binding to nonspecific antigen sites. However, the boar proacrosin antibodies reacts with only two bands (Mr 53 000 and 55 000) in the crude sperm extracts (Fig. 5a) which correspond to proacrosin (Figs. 2 and 4). In summary, we have developed polyclonal antibodies to highly purified human and boar proacrosin and have characterized the main components of the proacrosin-acrosin system which react with these antibodies. This is the first step in using these antibodies as a meaningful tool to analyze this important enzyme system. This, in turn, could enhance our understanding of physiological changes occurring in sperm prior to fertilization. Acknowledgements We thank Ms. Cynthia Bahr and Ms. Bonnie Baskett for typing this manuscript. We would also like to thank Dr. Bruce Lessley for his suggestions and help concerning the production of proacrosin antibodies. This research was supported by National Institute of Health Grants HD-09422 and HD-12863. References Kennedy, W.P., Parrish, R.F. and Polakoski, K.L. (1981) Improved method for the preparation and purification of boar m,,-acrosin. Biol. Reprod. 25, 197-201. Kilgore, L.L., Patterson, B.W. and Fisher, W.R. (1986) Immunologic comparison of the conformations of apolipoprotein B. J. Biol. Chem. 261, 8842-8848. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680--685. Lane, L.C. (1978) A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal. Biochem. 86, 655-664. Polakoski, K.L. and Parrish, R.F. (1977) Boar proacrosin: Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. J. Biol. Chem. 252, 1888-1894, Polakoski, K.L. and Siegel, M.S. (1986) The proacrosin-acrosin system. In: Andrology Male Fertility and Sterility (Paulson, J.D., Negro-Vilar, A., Lucena, E. and Martini, L. eds.), pp. 359-375. Academic Press, New York. Siegel, M.S., Bechtold, D.S., Kopta, C.I. and Polakoski, K.L. (1986) The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system. Biochim. Biophys. Acta 883, 567-573. Towbin, H., Staemell, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some application. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197-203.
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Elsevier Scientific Publishers Ireland Ltd.
JRI 00490
Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems Mark S. Siegel, Dana S. Bechtold, Janet L. Willand and Kenneth L. Polakoski Department of Obstetrics and Gynecology, Washington Universilty, St. Louis, Missouri (U.S.A.) (Accepted for publication 29 May 1987)
Summary Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55 000), alpha-acrosin (45 000-49 000) and beta-acrosin (34 000-38 000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological ditterences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar
Correspondence to: Kenneth L. Polakoski, Department of Obstetrics and Gynecology, Washington University, 4911 Barnes Hospital Plaza, St. Louis, MO 63110, U.S.A. 0165-0378/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
308
proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34 000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm. Key words: proacrosin, acrosin, proteinase, immunology, spermatozoa.
Introduction
Acrosin (EC 3.4.21.10) is a sperm proteinase with trypsin-like specificity that is believed to have a critical role in events leading to fertilization. Proacrosin is a zymogen precursor to acrosin and is the form that accounts for the majority of the acrosin that is present in epididymal and freshly ejaculated sperm. From studies on proteins isolated from boar sperm, a model system has been devised which assists in the design of investigations concerning the numerous components of the proacrosinacrosin system (reviewed by Polakoski and Siegel, 1986). The porcine species has been utilized for many of these studies because large amounts of material are available and the individual components are obtained in relatively stable forms. We have recently purified proacrosin from human sperm (Siegel et al., 1986). The partial biochemical characterization of this key protein has resulted in the observation of a number of similarities between the human and boar proacrosin-acrosin systems. These include similar molecular weights and interrelationships between the different forms of proacrosin and acrosin. Even though considerable progress has been made regarding the biochemical characterization of the proacrosin-acrosin system, little immunological information on any of its components is available. Furthermore, there are presently no reports on antibodies developed specifically to proacrosin. This is particularly surprising since immunochemical approaches have proven increasingly useful in probing the structural features of proteins (Kilgore et al., 1986) which may be particularly important in the regulation of this critical enzyme system. In the present investigation, polyclonal antibodies were used to provide a generalized view of the immunochemical identities of human and boar proacrosin. The data obtained are of particular importance because they
309
clearly demonstrate that there are immunochemicai differences between these species. These differences may be of particular interest in investigating the regulatory components of this system as well as in the design of immunochemical approaches to contraception. Materials and Methods
Reagents N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), benzamidine, dithioerythritol, dithiothreitol (DTT), Freund's incomplete adjuvant, 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA) and iodoacetamide were obtained from Sigma, St. Louis, MO. Glycine, Tris(hydroxymethyl)aminomethane (Tris), polyoxethylene sorbitol (Tween 20), acrylamide, 4-chloro-1-naphthol (HRP color reagent) and goat anti-rabbit horseradish peroxida~e conjugate were purchased from Biorad, Richmond, CA. Sodium dodecyl sulfate (SDS) and guanidine-HC1 were obtained from Pierce, Rockford, IL and ultrapure ammonium sulfate from Schwarz-Mann Inc, Spring Valley, NY.
Preparation and purification of human and boar proacrosin Human sperm were obtained from freshly ejaculated semen samples. The sperm were washed, acid extracted and proacrosin was purified as previously described (Siegel et al., 1986). Boar sperm were obtained by flushing the sperm through the caudae epididymidis with Hepes buffer (0.1 M) which contained benzamidine (0.05 M) at pH 7.5. The sperm were then washed free of epididymal fluid by centrifugation through 0.05 M benzamidine in 1.0 M sucrose (pH 6.0). The washed sperm were acid extracted and the proacrosin was partially purified via a pH 6.0 and then a 35% ammonium sulfate precipitation according to the procedures of Polakoski and Parrish (1977) as modified by Kennedy et al. (1981). The ammonium sulfate precipitated material was then further purified via a Superose 12 gel filtration procedure using a Pharmacia FPLC system as described for human proacrosin (Siegel et al., 1986). The peak fractions of proacrosin were dialyzed, pooled and used for the activation and immunoblotting experiments that are described below. However, to prevent proacrosin autoconversion from occurring during the antibody development, the samples that were used for raising the antibodies were treated in a slightly different manner prior to the Superose-12 gel filtration. For these studies each of the human and boar preparations were first reduced and then alkylated by a modification of the procedure described by Lane (1978). Briefly, the samples wcrc adjusted to a final concentration of 0.3 mg/ml in 6 M guanidinc-HCI at pH
310
2.5. The proteins were then reduced by the addition of 1/50 vol. of fresh 0.25 M dithioerythritol, heated for 2 min at 100°C and then cooled to 22°C. One twentieth volume of 0.25 M iodoacetamide was then added to the sample and incubated at 50°C for 15 min. The sample was then dialyzed against 1000-fold excess of double distilled water and iyophilized. The material was then resuspended in 200 ~1 of 8 M guanidine in 0.5 M acetic acid (pH 2.5) and injected onto the column.
Electrophoresis of proacrosin and acrosin Two micrograms of the purified human and boar proacrosin were autoactivated for 0, 5, 15, and 60 min by adjusting the pH to 8.0 with Tris buffer (final concentration 0.05 M). The autoactivation was stopped by the addition of an equal volume of sample buffer containing 4% SDS, 20% glycerol, 0.5 M DTT and the solutions were boiled for 2 min. Duplicate aliquots of each of these preparations were electrophoresed on two adjacent 12.5% discontinuous polyacrylamide gels (Laemmli, 1970) using a Hoefer Electrophoresis apparatus. Following electrophoresis for 3.5 h at 20 mA/gel, one of the gels was stained for protein using a silver staining procedure (Wray et al., 1981) so that the molecular weights of the proacrosin and acrosin could be determined. Proteins from the duplicate gel were transblotted and the resulting blots treated as described in the western blotting section below.
Production of human and boar proacrosin antibody Female New Zealand white rabbits were injected intradermally on day 0 and at two more times at 2-week intervals with either the purified human or boar proacrosin which was reduced and alkylated as described above. The proacrosin preparations (50 ~g) were mixed with equal volumes of Freund's incomplete adjuvant prior to injection. On the 9th day following the last injection, 50 ml of whole blood was collected from the rabbit ear vein, allowed to coagulate at room temperature for 4 h and the blood sera was then obtained by centrifugation of the blood at 10 000 x g for 10 min. The antibodies were obtained from immunized and pre-immunized sera and partially purified by a 40% ammonium sulfate precipitation for 1 h at 4°C. The samples were centrifuged and the resulting pellet was resuspended in 0.01 M Tris at pH 7.5 and subsequently dialyzed against the same buffer to remove the salts.
Western blotting Immunoblotting was performed with a slight modification of the procedure of Towbin et al. (1979). For these experiments, the proteins were electrotransferred at 4°C in a 0.03 M MES, 0.19 M glycine buffer (pH
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6.1) for 16 h at 30 V and then for 2 h at 50 V. The sheets were incubated for 2 h with the primary antibody preparation diluted either 1 : 25 (antihuman proacrosin) or 1 : 100 (anti-boar proacrosin) in the washing buffer containing 1% BSA. Preliminary experiments showed that these were the optimal concentrations for each of the primary antibodies. Results
The SDS-PAGE analysis of proacrosin and its activation products have been individually described for both the human (Siegel et al., 1986) and boar (Polakoski and Parrish, 1977). The results shown in Figs. 1 and 2 are
~,i,i~,~~,i~,i~,~i~,i,i,~,i,~,i i,,,i¸i~i,i~,i,i~!,i~,!~i~ IIIIIIIIII--BSA
PRO-ALPHA-
--A LD BETA---CA
-CYT A
B
C
C
D
Fig. 1. SDS-polyacrylamide gel electrophoretic analysis of the time course of human proacrosin autoconversion to acrosin. Equal volumes containing 2 Ixg of purified proacrosin and 0.1 M Tris (pH 8.0) were incubated for 0rain (lane A), 5 rain (lane B), 15 rain (lane C) and 60min (lane D). The reaction was stopped by the addition of 2% SDS/0.25 M dithiothreitoi and incubated for 2 min at 100°C. The samples were subjected to SDS-PAGE and the gels were silver stained for protein as described in Materials and Methods. The proacrosin (PRO), alpha-acrosin (ALPHA) and beta-acrosin (BETA) migrated as indicated. The standard proteins were (BSA, Mr, = 68 000), aldolase (ALD, M, = 40 000), carbonic anhydrase (CA, M, = 29 000) and cytochrome c (CYT C, M, = 12 800).
312
PRO-~ ALPHA-
-BSA i
BETA-
m ==
-ALD m
-CA
-CYT A
B
C
C
D
Fig. 2. SDS-polyacrylamide gel electrophoretic analysis of the time course of boar proacrosin autoconversion to acrosin. Purified boar proacrosin was autoactivated for 0 min (lane A), 5 min (lane B), 15 rain (lane C) and 60 min (lane D). Two micrograms of protein per lane were electrophoresed and the gels processed as described in the legend to Fig. 1.
presented in order to directly compare the major components of the proacrosin-acrosin system between these species and to demonstrate the relative amounts of each form of acrosin that is analysed via immunoblotting in Figs. 3 and 4. Proacrosin autoconversion The results presented in Fig. 1, lane A, show that human proacrosin has an apparent molecular weight between 51 000 and 55 000. When this preparation is autoactivated under the conditions described, most of the proacrosin is converted to alpha-acrosin (Mr --49 000) within 5 min (lane B). During the subsequent incubation, beta-acrosin is detected and becomes the predominant form at 60 min.
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H B
H
B
H
B
H
B
-BSA -ALD -CA
-CYT C
A
B
C
D
E
F
G
H
Fig. 3. lmmunoreaction of antibodies to human proacrosin towards the purified human and boar proacrosin-acrosin systems as determined by immunoblotting. Purified human (H) and boar (B) proacrosin were autoactivated for 0 rain (lane A and B), 5 min (lane C and D), 15 min (lane E and F) and 60 min (lane G and H) as described in the legend to Fig. 1. The samples (2 i~g/lane) were resolved by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose and probed with antibodies to human proacrosin as described under Materials and Methods.
The purified boar proacrosin activation profile (Fig. 2) was very similar to that described for human proacrosin. Although not easily observed, two bands of protein (M, = 53 000 and 55 000) were present in the highly purified boar proacrosin preparation (Fig. 2, lane A). Following proacrosin autoconversion for 5 min, apparently all of the proacrosin is converted to alpha-acrosin (M, = 45 000) (Fig. 2, lane B). After 60 min, most of the alpha-acrosin was converted to beta-acrosin (Fig. 2, lane D, .Mr,= 38 000).
314
H
B
H
B
H
B
H
B
--9SA
m
lid
--ALD
-CA
--CYT C
A
B
C
D
E
F
G
H
Fig. 4. Immunoblot of human and boar proacrosin and acrosin with antibodies to boar proacrosin. Human (H) and boar (B) proacrosin were autoconverted for various time intervals and analyzed for their immunoreactivity towards antibodies to boar proacrosin as described in the legend to Fig. 3.
Immunobloning An immunobiotting technique was utilized to analyse the specificity and sensitivity of rabbit antibodies directed to highly purified human and boar proacrosin. To determine the extent to which non-specific binding affects the immunoblotting procedure, control experiments were performed (in the absence of the primary antibody) with ammonium sulfate precipitated antibody from pre-immunized rabbit sera. In each of these cases there was no observed color reaction on the nitrocellulose sheets (results not shown) indicating that the reaction observed was not due to either a non-specific antibody interaction or to non-specific binding of the second antibody. The antisera to the human proacrosin was tested for its immunoreactivity with human proacrosin and acrosin as well as with the boar proacrosin and acrosin (Fig. 3). The human proacrosin antibody reacted with human proacrosin (lane A) as well as human alpha-acrosin (lanes B,
315
E and G). However, there was apparently no reactivity with the human beta-acrosin (lanes E and G). In addition, boar proacrosin (lane B) and to a lesser extent alpha-acrosin (lanes D and F) cross-reacted with the human proacrosin antibody (1 : 25 dilution), while the beta-acrosin did not (lanes D and F). The antibody to boar proacrosin (1 : 100 dilution) was also tested for its immunoreactivity with the human and boar proacrosin and acrosin (Fig. 4). There was no cross-reactivity observed between the human proacrosin, alpha-acrosin or beta-acrosin and the boar proacrosin antibody (lanes A, C, E and G) even at antibody dilutions of either 1:25 or 1:10 (results not shown). However, there was a strong immunoreaction between the boar proacrosin antibody and boar proacrosin (lane B) and
H
B
--ALD
-CA ii~ii~!~ ~,~i~~,~,~i~i~~
~ii~i~i i~!~~iI~i ~, ~ ~ ~
--CYT C A
B
316
H
B
H
B
-BSA -ALD -CA
-CYT
A
B
C
C
D
Fig. 5. (a) SDS-polyacrylamide gel electrophoretic analysis of non-purified acid extracts of human and boar sperm. Approximately 2 I~g of protein from non-purified human (lane A) and boar (lane B) sperm extracts were individually subjected to SDS-polyacrylamide get electrophoresis and the gels were silver stained for protein as described in Materials and Methods. (b) Immunoblot of unpurified human (H) and boar (B) sperm extracts using antibodies to highly purified human and boar proacrosin. The results of the immunoreaction of antibodies to human proacrosin against the sperm extracts (2 I~g/iane) are shown in lanes A and B. The immunoreaction of antibodies to boar proacrosin against the sperm extracts (2 p,g/lane) are shown in lanes C and D.
boar alpha-acrosin (lane D and F). A distinct but weaker reaction was observed for the boar beta-acrosin (lane H). Non-purified human and boar sperm extracts were used to determine if the antibodies reacted with other sperm antigens. In these preparations at least 95% of the total acrosin was present in the proacrosin form (results not shown). The human sperm extracts (Fig. 5a, lane A) were reacted with antibody to human proacrosin. The results shown in Fig. 5b (lane A)
317
demonstrate that the antibodies apparently reacted with 53 000, 49 000 and 34000 molecular weight antigens. Interestingly, several immunoreactive bands were detected at molecular weights above 68 000. However, when the boar sperm extracts (Fig. 5a, lane B) were reacted with the antibody to human proacrosin there were only two weakly immunoreactive bands (Mr = 55 000 and 53 000) observed. The boar proacrosin antibodies were also incubated with the nonpurified human and boar sperm extracts which had been electroblotted to nitrocellulose sheets. There was no cross-reactivity observed with the human sperm extract (Fig. 5b, lane C) while intense bands (Mr = 55 000 and 53 000) were observed in the boar extracts (Fig. 5b, lane D). Discussion
This report documents that: (a) in the human and boar proacrosinacrosin system there are similarities in the molecular weights of the main components as well as similar time courses for proacrosin auto-conversion; (b) antibodies can be produced against highly purified human and boar proacrosin; (c) human proacrosin antibodies that were developed cross-react with human proacrosin and alpha-acrosin while the boar proacrosin antibodies cross-react with boar proacrosin, alpha- and betaacrosin; (d) human proacrosin antibodies cross-react with boar proacrosin while the boar proacrosin antibodies do not react with the human proacrosin-acrosin system; (e) the antibodies that were developed were also shown to be highly specific for the proacrosin-acrosin system within each of the non-purified sperm extracts. As observed in Figs. 1 and 2, the molecular weights of the human and boar proacrosin-acrosin system are quite similar. The relative molecular weight for proacrosin is in the 51 000-55 000 range while alpha-acrosin is between 45 000 and 49 000 and beta-acrosin is about 34 000-38 000. In addition, the time sequence of autoconversion of proacrosin to acrosin was very similar in both species. Under the conditions employed, proacrosin conversion to alpha-acrosin occurred in less than 5 min while beta-acrosin was the predominant molecular weight form after 60 min incubation. Similar molecular weights and autoconversion times have been previously reported for the proacrosin-acrosin system from human (Siegel et al., 1986) and boar (Polakoski et al., 1977) sperm. However, the demonstration of the relative concentration of proacrosin and each of the forms of active acrosin at the different time points was required to estimate the cross-reactivity of the proacrosin antibodies in the western blotting experiments in Figs. 3 and 4. Although antibodies to acrosin from a variety of species have been
318
developed (reviewed by Polakoski and Siegel, 1986), no reports of proacrosin antibodies are available. The difficulty in making antibodies to proacrosin involved the probable autoconversion of proacrosin to active acrosin forms during the immunization process. This problem was alleviated by first reducing and then alkylating the proacrosin prior to the immunization thereby preventing proacrosin autoconversion. The ability of the proacrosin antibodies to cross-react with the specific components of the proacrosin-acrosin system was evaluated in Figs. 3, 4 and 5a. Interestingly, the antibodies to human proacrosin react with human proacrosin and alpha-acrosin but not with beta-acrosin (Fig. 3). This indicates that the antibodies that were raised against human proacrosin are directed to antigens or conformations that are lost during the final proteolytic conversion process which occurs during the formation of beta-acrosin from alpha-acrosin. This does not appear to be the case for the boar proacrosin antibodies since at least some of these react with the boar beta-acrosin (Fig. 4). It is not presently known if this is the result of significant differences in the structure of proacrosin from these two species. Notable differences exist with respect to the immune response of the human proacrosin antibodies with boar proacrosin and acrosin as compared to the boar proacrosin antibodies reactivity with human proacrosin and acrosin. The human proacrosin antibodies reacted predominantly with boar proacrosin, very slightly with the alpha-acrosin and not with beta-acrosin. The boar proacrosin antibodies, however, do not cross-react with any of the major components of the human proacrosin-acrosin system even at antibody concentrations ten-fold higher than these used in Fig. 4. It is possible that there are specific antigenic sites on human proacrosin-acrosin which are not common to the boar proacrosin-acrosin system. This, therefore, would permit an immunoreaction with human proacrosin antibody and human proacrosin but not the boar proacrosin antibody with human proacrosin. We are presently attempting to purify sufficient material from each of these proacrosin-acrosin systems to sequence these proteins so that it may be determined if the primary structural differences in these molecules could account for the observed immunological differences. The specificity of antibodies produced to the proacrosin-acrosin system are shown in Fig. 5a and b. The vast molecular weight range of proteins present in human and boar sperm extracts is shown in Fig. 5a. Despite the large numbers of proteins present, the major antigens that react with the antibodies to human proacrosin have relative molecular weights of 53 000-55 000 (Fig. 5b). The results shown in Figs. 1 and 3 strongly suggest that these antigens are proacrosin. However, minor
319
bands of reactivity were also observed in regions that correspond to molecular weights of 68 000 and above as well as at 49 000 and 34 000. It is not presently known if these molecular weight forms are part of the human proacrosin-acrosin system or represent possible binding to nonspecific antigen sites. However, the boar proacrosin antibodies reacts with only two bands (Mr 53 000 and 55 000) in the crude sperm extracts (Fig. 5a) which correspond to proacrosin (Figs. 2 and 4). In summary, we have developed polyclonal antibodies to highly purified human and boar proacrosin and have characterized the main components of the proacrosin-acrosin system which react with these antibodies. This is the first step in using these antibodies as a meaningful tool to analyze this important enzyme system. This, in turn, could enhance our understanding of physiological changes occurring in sperm prior to fertilization. Acknowledgements We thank Ms. Cynthia Bahr and Ms. Bonnie Baskett for typing this manuscript. We would also like to thank Dr. Bruce Lessley for his suggestions and help concerning the production of proacrosin antibodies. This research was supported by National Institute of Health Grants HD-09422 and HD-12863. References Kennedy, W.P., Parrish, R.F. and Polakoski, K.L. (1981) Improved method for the preparation and purification of boar m,,-acrosin. Biol. Reprod. 25, 197-201. Kilgore, L.L., Patterson, B.W. and Fisher, W.R. (1986) Immunologic comparison of the conformations of apolipoprotein B. J. Biol. Chem. 261, 8842-8848. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680--685. Lane, L.C. (1978) A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal. Biochem. 86, 655-664. Polakoski, K.L. and Parrish, R.F. (1977) Boar proacrosin: Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. J. Biol. Chem. 252, 1888-1894, Polakoski, K.L. and Siegel, M.S. (1986) The proacrosin-acrosin system. In: Andrology Male Fertility and Sterility (Paulson, J.D., Negro-Vilar, A., Lucena, E. and Martini, L. eds.), pp. 359-375. Academic Press, New York. Siegel, M.S., Bechtold, D.S., Kopta, C.I. and Polakoski, K.L. (1986) The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system. Biochim. Biophys. Acta 883, 567-573. Towbin, H., Staemell, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some application. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197-203.