Hum Genet (1991) 87:635-641

9 Springer-Verlag1991

Review article

Acrosin, the peculiar sperm-specific serine protease Uwe Klemm I, Werner Miiller-Esterl 2, and Wolfgang Engel 1 l Institut for Humangenetikder Universitfit, Gosslerstrasse 12d, W-3400 G6ttingen, Federal Republic of Germany 2Institut for PhysiologischeChemie und Pathobiochemieder Universit~it,Duesbergweg6, W-6500 Mainz, Federal Republic of Germany Received January 8, 1991 / Revised April 10, 1991

Summary. The sperm enzyme acrosin has long been known as one of the key enzymes in the mammalian fertilization process. Elucidation of primary structures of preproacrosin from various species have allowed a deeper insight into the structural organization and the complex evolution of the sperm proteinase acrosin. In addition to the typical elements of serine proteases, the acrosin molecule possesses one novel domain that might convey DNA-binding properties.

Introduction The long history of the study of proteolytic enzymes of the sperm cell was initiated in 1935 by a report of Yamane (Yamane 1935), describing the solubilizing effect of rabbit sperm extracts on the outer investments of oocytes. These findings stimulated the isolation and purification of sperm enzymes in the following decades. Many investigators focused on the major component of the acrosomal content, a trypsin-like enzyme called acrosin (EC 3.4.21.10) (Zaneveld et al. 1971). Following detailed analysis with natural and artifical substrates and inhibitors (Stambaugh and Buckley 1968), acrosin was classified as a member of the serine protease superfamily. This classification was supported by limited amino acid sequence information from boar (Fock-Ntizel et al. 1980, 1984) and goat acrosin (Hardy et al. 1989) obtained by the classical Edmann degradation method. Using the methods of recombinant DNA technology, difficulties in protein sequencing were bypassed and the primary structures for human (Baba et al. 1989a; Adham et al. 1990), boar (Adham et al. 1989; Baba et al. 1989b), mouse (Klemm et al. 1990; Kashiwabara et al. 1990a) and rat (Flake 1990; Gerloff 1990) acrosin could be predicted from their corresponding nucleotide sequences. Sequence comparison with other members of the serine protease superfamily have allowed the identification of a novel domain unique to the acrosin-type of proteases; Offprint requests to." W. Engel

this domain might excecute key functions of acrosin unrelated to its proteolytic capacity.

Localization of sperm acrosin The sperm protease, acrosin, is contained by the acrosome, a modified lysosome at the apical end of the spermatozoon. The affinity of acrosin to artifical and natural membranes (Brown and Hartree 1976; Parrish et al. 1978; Straus et al. 1981) plus the capacity of phospholipid vesicles to promote the autoactivation of the zymogen proacrosin (Parrish et al. 1978) indicate an association of the protease with the acrosomal membranes. Further evidence for this topography has been given by the immunohistochemical localization of acrosin activity at the surface of acrosome-reacted spermatozoa, implying the attachment of acrosin to the inner acrosomal membrane (Schill and Wolff 1974; Green and Hockaday 1978). However, hydropathy plots of the available acrosin protein sequences do not support such a hypothesis (data not shown). Two stretches with a hydrophobic character are notable in mammalian acrosin sequences, one at the amino-terminus representing the putative signal peptide, and another in the vincinity of the active site (serine) forming part of the catalytic charge relay system. The former hydrophobic segment is cleaved off before acrosin enters the acrosome, whereas the latter is also present in other serine proteases not associated with biological membranes. Thus, the structural basis of the interaction of acrosin with biological membranes, a prerequisite for zona binding and penetration (T/Spfer-Petersen 1988), remains to be explained.

Transcriptional and translational activity of mammalian acrosin genes during spermatogenesis Since the genome of spermatozoa is transcriptionally inactive because of the composition and compaction of the chromatin, acrosin mRNA must be synthesized at earlier stages of spermatogenesis. Stage-specific cell types reco-

636 vered from bovine testis have been used in Northern blotting experiments to demonstrate that the first acrosin transcripts are detectable in early round spermatids (Adham et al. 1989). In situ hybridization performed on testis sections of different mammalian species has shown that acrosin mRNAs are equally abundant in haploid round spermatids and elongating spermatids (Adham et al. 1989, 1990; Klemm et al. 1990). The combined data from our Northern blotting experiments and in situ hybridization studies clearly indicate, in agreement with recently published data from Kashiwabara et al. (1990a), the acrosin gene activity is first found in the postmeiotic stages of spermatogenesis. However, haploid gene expression has been described in the mouse (Kashiwabara et al. 1990b). Immunocytochemical analysis using polyclonal antibodies directed against boar acrosin has enabled the temporal appearance of acrosin/proacrosin to be assigned to the stages of early round spermatids (F16rke et al. 1983; F16rke-Gerloff et al. 1983; Arboleda and Gerton 1988). These findings, obtained in various mammalian species including man, contradict results of studies of acrosin biosynthesis in mouse, using a monoclonal antibody; these studies pinpoint the initiation of acrosin biosynthesis to the last phase of the spermatogenic cycle, i.e., to elongating step-9 mouse spermatids (Kallajoki et al. 1986). In view of the fact that acrosin esterase activity is detectable in early round spermatids enriched from a bovine testis suspension (Mansouri et al. 1983), one is tempted to suggest that the observations of Kallajoki et al. (1986) are attributable to post-translational modifications of the acrosin molecule, occurring during spermatid elongation and recognized by the monoclonal antibody.

Molecular architecture o f m a m m a l i a n acrosin

The accumulation of data concerning the primary structure of acrosin enables us to elaborate a general structural model for the mammalian molecule comprising three major domains, i.e., the zymogen domain, followed by the catalytic domain and the tail domain (Fig. 1). The amino-terminal zymogen domain and the catalytic domain are each characterized by a striking sequence identity among acrosin molecules from various species and between the members of the serine protease superfamfly, whereas the carboxy-terminal tail domain is rich in

5/6

proline, a feature that is unique to the acrosin-type of proteases and that is not present in other members of the superfamily. The tail domain differs from the other two acrosin domains in that its structure is highly variable among the acrosin molecules from diverse species.

Zymogen domain

All of the proacrosin sequences elucidated so far contain a signal sequence of 15-18 amino acid residues (Fig. 2); the enzyme is synthesized as a preproenzyme, preproacrosin. Signal sequences reflect a characteristic 15-25 amino acid hydrophobic extension of the NHz-terminus of proteins destined for export (von Heijne 1983, 1984). Once initiated, signal sequences are cleaved from the growing protein chain at specific sites during the translocation process through the membranes of the rough endoplasmic reticulum (yon Heijne 1983, 1984). Small neutral amino acid residues at positions - 3 and - 1 are required for acceptable cleavage sites, e.g., valine or alanine ( - 3 ) and alanine ( - 1 ) (von Heijne 1984). They are found in typical positions of mammalian acrosin signal sequences, suggesting that canonical signal peptides are involved in the first step of transport to their site of storage, i.e., the acrosome. The signal peptide is followed by a short strech of 23 amino acid residues representing the "activation peptide" of the zymogen. Positions 1-23 of the mature boar preproacrosin sequence determined in our group are identical with the sequence of the previously determined 4.2-kDa boar acrosin light chain (Fock-Ntizel et al. 1984), and positions 24-74 correspond to the previously published amino-terminal part of the 37-kDa heavy chain (Fock-Ntizel et al. 1980; Fridberger et al. 1984). Inspection of the complete preproacrosin primary structure demonstrates that the activation of the proteolytically inactive proacrosin molecule to the active enzyme acrosin proceeds via cleavage of the peptide bond Arg-23-Val-24 without loss of amino acid residues from the newly generated termini of the heavy and light chains. Autoactivation (Polakoski and Parrish 1977) may be one of the critical steps initiating conversion of the singlechain proacrosin (Gilboa et al. 1973; Cechova et al. 1988) to the mature two-chain form of a-acrosin (Fock-Nazel et al. 1984). The conservation of Arg-23 observed in all species examined so far lends support to the postulated

2

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Iiiiiiii!iiii!iiiiiiiiiiiiiliiiiiiiiiiiiiiiiiililililiiiiiiiit I /

Zymogen domain

Catalytic domain

Tail domain

Fig. 1. General architecture of mammalian preproacrosin. The

three domains are drawn schematically: the zymogen domain including the signal peptide and the light chain, the catalytic domain representing the amino-terminal part of the heavy chain, and the tail domain corresponding to the carboxy-terminal part of the

heavy chain. The putative arrangement of disulfide loops (nos. 16) is shown (data obtained from Young et al. 1978). Localization

of the catalytic triad (His-70, Asp-124 and Ser-223) is indicated by asterisks

637 signal ha pa ma ra ba

I VGGKAAQHGAWPWMVSLQZFTY-NSHRYHTCGGSLLN •••MLPTAvL•VLAv•vAARDNATCDGPCGLRFRQKLEsGMRvvGGM•AEPGAWPWMvSLQIFMYHNNRRYHTCGGILLN ---MLPTAFWSVK-vsAGAKDNATCFGPCGLRTRQNSQAGTRIVSGQsAHvGAWPWMvSLQIFTsHNSRRYHACGGsLLN MvEMLPTVvALvLAVSvvAKDNTTCDGPCGLRFRQNPQAGIRIVGGQTSSRWAWPWMvSLQIFTS•NSRRYHACGGsLLN MLPT

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328 326 326 330

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tail domain . . . . . . . . . . . . . . . . . . . . . . . . . . :::::: ::::::::::::::::::::::: : ~:~-;-;-;-:-:~-;-x-;-;-:~+;-;-x-.x-x.-x. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V G IT S W G V G C A R A K R P G IY T A T W P Y L N W I A S K IG S N A L R M I Q S A T P P P P T T R P P P I - R P P F S H P I S A H L P W Y F Q P P P R P L P P R P P A A Q P R V G I T S W G V G C A R A K R P G V Y T S T W P Y L N W I A S K I G S N A L Q M V Q L G T P P R P S T P A P P V - RPP- S V Q T P V R P P W Y F Q R P P G P - - S Q Q P G S R P R VG I T S W G V G C A R A K R P G V Y T A T W D Y L D W I A S K I G P N A L H L I P A A T P H P P T T R H P M V S F H P P S F . . . . R P P W Y F Q H L P S R P L Y L R P L R P L L V G I T S W G V G C A R A K R P G V Y T A T W D Y L D W I A S K I G P T A L H L I Q P A T P H P P T T Q Q P V I S F H P P STPP S L V L P T P V S S A A L P T P P R P L L H Q P S VG I T S W G V G C A R A K R P G V Y T S T W S Y L N W I A S K I G S N T V H M I Q L P T A P P A S T P A A Q A S PG SVQP-- S I R P P W F F Q H V P Q P P P S Q Q A I A V A Q VGITSWGVGCARAKRPG

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P•APPPAPPPPP•PP•P•PPPP•••P•••SA--KPP•ALS•AKRLQQLIE-LKGTGNSLVERSYYETETTDLRR-TARLLI HRPSSTQTSSSLMPLLSPPTPAQPSFTIATQHMRHRTTLSFARRLQRLIEALKMRTYPM-K--YPPSTvDKELP-LPLLHv SVHTSSAPvIPLLsLLTPVQPvSFTLAAYHTR--HHTTLSFASALQHLIEALKMRTYPI-K--YPPvQWTKELPAP-LLHv PSPSLKPPALSPTWPPDPPRPPPPQPS--T-R--PPQALSFAKRLQQLIEvLKGKTFLNEKSNYE-METTGPSRTTALLLI P

LSFA

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Fig. 2. Comparison of the amino acid sequences of human, boar, mouse, rat and bovine preproacrosin as deduced from their nucleotide sequences. The primary structures of human (ha), porcine (pa), mouse (ma), and rat preproacrosin (ra) are derived from Adham et al. (1990, 1989), Klemm et al. (1990), Flake (1990) and Gerloff (1990). The partial primary structure of bovine acrosin (ba) is also included (unpublished results). The relative positions of the signal peptide, the light chain, the heavy chain and the amino-terminal part of the tail domain are indicated above the sequences. Potential N-linked glycosylation sites are marked by asterisks. Residues of the catalytic triad (His-70, Asp-124, and Ser223) are denoted by arrows

autocatalytic mechanism, since the endopeptidase acrosin has a strong preference for arginine bonds (Polakoski and McRorie 1973). Furthermore, human, rat and mouse acrosin match the boar acrosin light chain sequence in 74%, 69% and 65% of residues, respectively, suggesting a common activation mechanism for mammalian acrosins. Release of an amino-terminal "activation peptide" is often found in the activation cascades of mammalian serine proteases, i.e., in the processing of bovine pro-

Y

L

thrombin (MacGillivray and Davie 1984) and bovine coagulation factor X (Titani et al. 1975). In the case of acrosin, the propart of the zymogen remains attached to the catalytic units via two disulfide bridges connecting the heavy and light chains of the mature acrosin. The two cystine residues of the light chain (position 6 and 10, respectively) are invariant among the m a m m a l i a n acrosin sequences. Reverse-phase H P L C has recently demonstrated that Cys-6 and Cys-10 on the boar acrosin light chain are covalently linked by two interchain disulfide bonds to Cys-136 and Cys-144, respectively, on the heavy chain (T6pfer-Petersen et al. 1990). The carbohydrate attachment site in position 3 exhibiting the typical acceptor sequence of Asn-Xaa-Thr (Pless and Lennarz 1977) is present in all studied m a m m a l i a n preproacrosin molecules (Fig. 2). In boar, glycosylation of Asn-3 has been verified by biochemical analysis (Fock-Ntizel et al. 1984). Another possible carbohydrate attachment site of AsnXaa-Thr, conserved in all acrosin molecules investigated so far, is at position 195-197. Other serine proteases, i.e., bovine plasminogen (Schaller et al. 1985), also exhibit two carbohydrate moieties. In the mammalian acro-

638 sin/proacrosin system, glycosylation seems to be a general post-translational modification, since the carbohydrate component ranges between 3% in ram acrosin (Brown and Hartree 1978) and 8% in rabbit (Mukerji and Meizel 1979) and boar acrosin (Mtiller-Esterl and Fritz 1980) by weight.

Catalytic domain The heavy chains of mammalian acrosin contain all of the functional elements identified in the active sites of serine proteases, i.e., the histidine, aspartic acid and serine residues (Fig. 2). The acrosin heavy chain shows great similarity between the various species. As in the light chains, all of the cystine residues (n = 10) are conserved in the heavy chains of the various acrosin molecules (Fig. 2). A comparison with the primary structures of other serine proteases reveals that 8 out of the 12 cystine residues are conserved in the serine protease superfamily (data obtained from Young et al. 1978). Based on this similarity with other serine proteases, the intra-chain disulfide bonds of the acrosin molecules can be arranged as depicted in Fig. 1. The intra-chain bridges 1,3 and 4 adjacent to the active site elements have been conserved in all serine proteases over a period of about 1.5 billion years (see Young et al. 1978). Disulfide bond 2 is similar to an intramolecular disulfide bond that is only found in the closely related serine protease plasmin. The predicted arrangement of the disulfide bonds has been established by biochemical analysis for the boar acrosin molecule (T/Spfer-Petersen et al. 1990). The carbohydrate attachment site (position 195-197) mentioned earlier has also been verified by biochemical analysis (T6pfer-Petersen et al. 1990), although the exact nature of the oligosaccharide chain is still unknown.

Acrosin tail domain The predicted acrosin primary structures from various species show a remarkable degree of identity for the zymogen domain and the catalytic domain, whereas the sequence similarity drops sharply in the tail domain of 125-128 residues (see Fig. 1). Within the serine protease family, an COOH-terminal extension of the catalytic domain is unique to the acrosin-type enzymes, leading to the assumption that the tail domain might be of critical importance to the function of the enzyme in the mammalian fertilization process. Furthermore, an unusually high content in proline ranging between 18% (mouse) to 34% (human) is not present in other mammalian serine proteases and points to an unusual spatial structure of the acrosin tail domain. Several attempts have been undertaken in the past to explain the possible biological role of the extra domain located at the extreme COOHend of acrosin-type enzymes. Liberation of the tail domain during maturation from a-acrosin into the f3-form has been reported by Zelezna and Cechova (1982). Because of its unusual amino acid composition (high con-

tent of prolines and of hydrophobic amino acid residues), the tail domain was thought to be responsible for the attachment of the acrosin molecule in or on the acrosomal membrane (Zelezna et al. 1989; Zelezna and Cechova 1982). However, we and others have been unable to identify, from hydropathy profiles of the predicted acrosin molecules, hydrophobic stretches required for integration into the acrosomal membrane (own unpublished data; Baba et al. 1989b). Since several authors have reported a lectin-like carbohydrate-binding activity, located on the acrosin heavy chain but not functionally coupled to the catalytic activity of the enzyme (Jones et al. 1988; T6pfer-Petersen and Henschen 1988), we expected to be able to identify such a region within the tail domain. However, sequence comparisons of acrosin with the available amino acid sequences of lectin-like proteins (data not shown) has failed to identify a putative lectin-like sequence. In the light of recent reports on the isolation and characterization of sperm proteins that are unrelated to acrosin but that bind to sperm receptors exposed by the zona pellucida of the ovum (for review see Wassarman 1988), the participation of acrosin in species-specific zona recognition and/or the binding process appears to be unlikely.

Hypothetical function of the acrosin tail domain Sequence comparison of acrosin tail domains with sequences available in the Swiss Prot and EMBL data bank shows a remarkable percentage of amino acid identity with several DNA-associated and/or DNA-binding proteins. Similarity to genes involved in gene regulation is not restricted to the amino-terminal part of the tail domains that are rich in proline; there is also a remarkable percentage of identity when comparing the highly conserved motif ( L S F A K R L Q Q L I E X L ) of the acrosin tail domains with sequences of proteins interacting with DNA. Most interestingly, one of the best characterized proteins involved in mammalian gene regulation, the CTF/NF-1 transcriptional activator, is also divided into two domains, a proline-rich transcriptional activator domain and, next to it, a DNA-binding domain (Mermod et al. 1989). One may hypothesize that the acrosin tail domains with a proline content of between 18% and 34% have the potential of forming similar spatial structures as proposed for the CTF/NF-1 proline-rich activator domain (25% of the residues are prolines) (Mitchell and Tijan 1989). In addition, the hypervariable acrosin tail domains exhibit one motif ( L S F A K R L Q Q L I E X L ) invariant in all species investigated so far. Because of its similarity to DNA-binding proteins, the motif may serve as a DNA-binding peptide with the role of simply positioning the basic proline-rich segment in order for it to interact with DNA (for review see Struhl 1989). Unfortunately, the L S F A K R L Q Q L I E X L motif does not match other well known DNA-binding motifs, such as the leucine zipper, in a satisfying manner. However, combining the findings of our computational analysis of the proline-rich acrosine tail domain with recently published data, one might hypothesize that, for example, liberation of this domain during acrosin mat-

639 uration (Zelezna et al. 1989) is a prerequisite for the transacting facility of the peptide. One might further speculate that the acrosin tail domain or fragments thereof play a role in governing normal embryonic development, in contrast to haploid parthenogenesis, which can occur without participation of the spermatozoon. Indeed, the activation of rabbit oocytes has recently been shown to be dependent on a factor obtained from rabbit spermatozoa (Stice and Robl 1990). In summary, we would like to present the cleavable proline-rich acrosine tail domain with its affinity for D N A (by computational analysis) as a possible candidate transforming the maternal oocyte program to the embryonic program.

Evolutionary origin of mammalian acrosin From the data pool of the available protein sequences, it is possible to derive an evolutionary tree showing the ancestral relationship of acrosin with the other descendants of the serine protease superfamily. Sequence identities for the catalytic domains are typically between 31.1% (boar acrosin vs. porcine elastase) and 37.3% (mouse acrosin vs. bovine plasminogen). The best sequence match has been found with the pancreatic enzyme trypsinogen (36.6% identity). Moreover, the remarkable identity of acrosin with the fibrinolytic enzyme plasminogen (37.3% identity) is striking, suggesting a more recent onset of divergent evolution. Figure 3 shows the evolutionary tree for the serine protease superfamily elaborated from the data obtained by amino acid alignment of several serine proteases versus mammalian acrosin by the D N A star* computer program, and from the derived evolutionary distances in PAMs (PAM = unit of Accepted Point Mutations; 1 PAM = one accepted amino acid mutation per 100 links of protein). The branch lengths showing the evolutionary distances were derived from the percentage of amino acid identity and the rate of mutation acceptance of 5.9 PAMs for the mammalian trypsin family Trypsin bovine Kallikrein porcin~.,~ ~0 / Elastase Plasmin 5 71 / /~hymotrypsin Factor

Thro

X

~ 59 \y 52 ~'~ 3 U

166 ~ / /

B

/

Trypsin

6~//bacterial

64

Fig. 3. Model for the evolutionary tree of serine proteases including the sperm-specific protease acrosin. Branch lengths given in PAMs are drawn in proportion to the number of mutations that have occurred between the related proteins during divergent evolution. Branch attachment sites were evaluated by changes in disulfide bridges and by the number of cystine, methionine and tryptophane positions. Gene duplications indicated by solid points are in accordance with the evolutionary tree elaborated by Young et al. (1978)

(Dayhoff 1978). The evolutionary distance, particularly the branch lengths, are in good agreement with earlier published data (Young et al. 1978), with the exception of the evolutionary distance between acrosin and bacterial trypsin. Our results clearly indicate that the acrosin protein sequence diverges with a similar rate of mutation as calculated for the trypsin family, and not at a three times faster rate as suggested by others (Hardy et al. 1989). Following the time scale for serine protease phylogeny established by Young and coworkers in 1978, it becomes obvious that the gene duplication generating the ancestral acrosin gene took place about 1 billion years ago. Most interestingly, scyphozoans and hydrozoans acquired a rudimentary acrosome about 1 billion years ago (Bacetti 1979), suggesting that acrosin and the acrosome evolved simultaneously. To date, the ascidian Halocynthia roretzi is the most primitive animal for which an acrosin-like enzyme has been reported (Sawada et al. 1984). Together with the enzyme spermosin, acrosin plays a pivotal role in the fertilization process of this species. Although both enzymes are serine proteases, they are distinguishable by their substrate specificity and affinity to artificial and natural inhibitors (Sawada et al. 1984). Even in man, where acrosin was generally believed to be the only trypsin-like enzyme, a second trypsin-like enzyme, called sperminogen (spermosin), is present (Siegel et al. 1987).

Organization of the proacrosin gene We recently reported the isolation and sequencing of the human proacrosin gene (Keime et al. 1990) and we can now support these findings by the sequencing data of the rat and mouse proacrosin gene (Flake 1990; Wilhelm 1990). The gene contains 6 exons and 5 introns, one of which is located in the 5' untranslated region. Since acrosin is accepted as a trypsin-like enzyme, the close relationship of the proacrosin gene to the trypsin and kallikrein gene is not surprising. As in other members of the serine protease superfamily, the coding sequence of the proacrosin gene is spread over 5 exons. Thus, exons E2, E3, and E5 code for the three active site residues (His69, Asp-123 and Ser-221) forming the charge relay system responsible for the hydrolytic capacity of the enzyme. Depending on the general mechanism of the catalytic triad exon-intron organization between the proteinase domains, elements of the catalytic triad of the different serine proteases seem to be very similar. Serine proteases, such as prothrombin or tissue plasminogen activator, which have acquired additional functional domains, show groups of exons upstream from the exons coding for the proteinase domain. However, the nonenzymatic part of the proacrosin molecule, the proline-rich tail domain, is encoded by a region located downstream from the exons coding for the acrosin proteinase domain. Furthermore, the codons for the acrosin tail domain are part of the 555-bp exon E5, which bears the sequence information for the Ser-221. In addition to its unusual amino acid distribution, the acrosin tail domain shows a localization within the proacrosin gene that is

640 u n i q u e in the serine p r o t e a s e superfamily. T h e s e results s u p p o r t o u r hypothesis of a novel f u n c t i o n of acrosin, in a d d i t i o n to its proteolytic activity in the fertilization process.

Conclusions and prospects P r e d i c t i o n of acrosin p r i m a r y structures from the corres p o n d i n g n u c l e o t i d e s e q u e n c e s has r e v e a l e d that acrosin is o n e of the ancestral serine proteases e n d o w e d with a signal p e p t i d e , a catalytic d o m a i n h o l d i n g the catalytic triad, and a tail d o m a i n that is u n i q u e a m o n g the m e m bers of the serine protease superfamily. Similarities bet w e e n the acrosin tail d o m a i n a n d D N A - b i n d i n g a n d / o r D N A - a s s o c i a t e d proteins, e.g., transcriptional activators, points to a specific role of the tail d o m a i n d u r i n g the m a m m a l i a n fertilization process. F u t u r e studies a i m e d at the e l u c i d a t i o n of the precise f u n c t i o n of the acrosin tail d o m a i n will take a d v a n t a g e of the availability of n u m e r ous m a m m a l i a n acrosin D N A s e q u e n c e s allowing the application of r e c o m b i n a n t D N A m e t h o d o l o g y . T a r g e t e d m u t a g e n e s i s of the D N A s e q u e n c e c o r r e s p o n d i n g to the tail d o m a i n via h o m o l o g o u s r e c o m b i n a t i o n p r o m i s e s to give d e e p e r insight into the biological role of this part of the acrosin molecule.

Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft (En 84/18-4; Mu 598/1-8).

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