Biocatalysis in Agricultural Biotechnology - American Chemical Society

Chapter 15

Structure—Function Properties of the Sperm Enzyme Acrosin Jerry L. Hedrick, Umbert A. Urch, and Daniel M . Hardy

Downloaded by RUTGERS UNIV on March 3, 2016 | http://pubs.acs.org Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch015

Department of Biochemistry and Biophysics, University of California, Davis, CA 95616 The sperm enzyme acrosin functions in sperm binding to, and penetration of, the egg zona pellucida. Acrosin is a three domain glycoenzyme, possessing a serine active site protease, a hydrophobic binding domain, and a recently described carbohydrate (fucose) binding domain; it is the first described lectin-protease. Its functional properties, including activation of a proenzyme as well as subsequent processing and separation of its three functional domains, are controlled via limited proteolysis (autolysis). Structure-function models for acrosin are presented. Functional membrane-bound forms of the lectin -protease are proposed which both hydrolyze and bind carbohydrate or just bind carbohydrate. Acrosin's properties provide for sperm binding and progressive penetration of the zona pellucida, essential cellular steps in the fertilization process. A l l animal eggs are surrounded by egg envelopes which must be traversed by sperm on their journey to the egg surface. These egg envelopes are known by various terms in different organisms, being called the vitelline layer in sea urchins, the vitelline envelope in amphibians, and the zona pellucida in mammals. The fertilizing sperm must penetrate the egg envelope in order to fuse with the plasma membrane and transfer its hap1οid set of chromosomes into the cell cytoplasm. Involvement of sperm proteolytic enzymes in fertilization processes has a long history. Perhaps the first definitive observation that sperm proteases affected egg envelopes was that of Tamane in 1935. He demonstrated that an extract of rabbit sperm dispersed the cumulus cells and solubilized the zona pellucida (ZP) or egg envelope of the rabbit egg (1). The presence of proteases in the sperm extract was presumed by analogy with the dispersing action of the trypsin activity in pancreatin. In 1939, Tyler obtained an extract from sperm of the giant keyhole limpet Meeathura crenulata which dissolved the egg envelope without affecting the egg itself. He coined the term "egg membrane lysin", which has since been 0097-6156/89/0389-0215$06.00/0 • 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, John R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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shortened to " l y s i n " , to describe those enzymes and factors in sperm which assist the sperm in penetrating the egg envelopes. The observations from Dan's laboratory in 1956 associated sperm l y s i n with the acrosome i n Mytilus ednlis sperm and i t s release or exposure during the sperm acrosome reaction. A f t e r the description of the acrosome reaction in mammalian sperm by Austin's laboratory in the late 1950's, studies on acrosomal proteases from mammalian sperm were i n i t i a t e d by several groups, f i r s t perhaps in Hartree's laboratory in Cambridge (for review see 2), but also by Stambaugh's lab in Philadelphia and Williams' group in Georgia. A f t e r intensive debate during the late 60's, the name acrosin was assigned to the t r y p s i n l i k e a c t i v i t y of the enzyme associated with the acrosome in mammalian sperm. The term l y s i n remains a generic term r e f e r r i n g to the enzyme or factors which s o l u b i l i z e or s p e c i f i c a l l y hydrolyze components of the egg envelopes, whereas acrosin is a s p e c i f i c or v a r i e t a l name for one p a r t i c u l a r enzyme which p r o t e o l y t i c a l l y hydrolyzes selected glycoproteins of the ZP. Non-enzymatic lys ins, discovered by HainoFukishima, bind stoichiometrically to envelope components thereby effecting their s o l u b i l i z a t i o n (for review see 3.)· However, this mechanism for sperm penetration of the egg envelope seems to be limited to marine mollusks. Contemporary studies on the ZP l y s i n acrosin were perhaps i n i t i a t e d with the observation that synthetic protease substrates were hydrolyzed by sperm extracts (4) and that the enzyme could be e f f e c t i v e l y extracted from sperm at pH 3 (5.). The enzyme has now been p u r i f i e d from several species, shown to be s p e c i f i c for Arg/Lys peptide bonds, and to be a serine active s i t e endoprotease. In 1972, Meizel provided evidence that a zymogen form of acrosin existed, termed proacrosin (6). Sperm hydrolases have often been suggested as being sperm l y s i n s based largely on circumstantial evidence, such as egg envelope d i s s o l u t i o n by heterogeneous sperm enzyme extracts. Envelope d i s s o l u t i o n by i t s e l f i s an i n s u f f i c i e n t c r i t e r i o n for a p a r t i c u l a r enzyme or complex of enzymes functioning as sperm l y s i n s . Criteria which must be met by sperm enzymes to q u a l i f y as sperm l y s i n s were suggested by Hoshi (3). We have modified the c r i t e r i a in view of recent observations by a number of investigators. C r i t e r i a which must be met to establish that a sperm enzyme is a l y s i n are: a) The active enzyme must be s p e c i a l l y and temporally located on the sperm so that i t i s in the right place at the r i g h t time to function in gamete interaction, b) The enzyme must s e l e c t i v e l y hydrolyze peptide bonds present i n the egg envelope glycoproteins, c) I n h i b i t i o n of the a c t i v i t y of the enzyme on the sperm surface must i n h i b i t egg envelope penetration, d) Appropriate chemical or immunological modification of the egg envelope glycoprotein, which is the substrate for the sperm l y s i n , w i l l prevent penetration of the egg envelope. No sperm enzymes have yet been described which meet a l l of these c r i t e r i a . However, acrosin s a t i s f a c t o r i l y meets the f i r s t three c r i t e r i a and, with the i d e n t i f i c a t i o n of the zona pellucida glycoprotein which i s a substrate for acrosin, i t i s now possible to approach experimentally the fourth c r i t e r i o n (2)·



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A v a r i e t y o f p r o t o c o l s have been used t o p u r i f y a c r o s i n from sperm of d i f f e r e n t s p e c i e s . Two approaches are commonly u s e d : a) p u r i f i c a t i o n o f the zymogen form, p r o a c r o s i n , from sperm e x t r a c t s f o l l o w e d by a u t o a c t i v a t i o n o f the p u r i f i e d zymogen and subsequent i s o l a t i o n of the a c t i v e enzyme, and b) p u r i f i c a t i o n o f the a c t i v e enzyme from sperm e x t r a c t s i n w h i c h p r o a c r o s i n a c t i v a t i o n has a l r e a d y o c c u r r e d . P r o p e r t i e s o f p r o a c r o s i n and a c r o s i n w h i c h have been e x p l o i t e d f o r purposes of p u r i f i c a t i o n i n c l u d e : a) r e s i s t a n c e t o i r r e v e r s i b l e d e n a t u r a t i o n by a c i d i c pH or by c h a o t r o p e s , b) h y d r o p h o b i c i t y , c) g l y c o s y l a t i o n , d) h i g h p i , and e) chemical s p e c i f i c i t y of the enzyme's s u b s t r a t e b i n d i n g s i t e . Since n e i t h e r p r o a c r o s i n a u t o a c t i v a t i o n nor p r o t e o l y s i s by a c r o s i n occurs a t a c i d i c pH, n e a r l y a l l a c r o s i n and p r o a c r o s i n p u r i f i c a t i o n methods use chromatography a t a pH l e s s than 4.5 f o r one o r more steps (8-13), thereby a v o i d i n g premature a u t o a c t i v a t i o n o f p r o a c r o s i n or l o s s of a c r o s i n a c t i v i t y due to a u t o l y s i s . Chaotropes such as u r e a and g u a n i d i n e - H C l have been used t o d i s r u p t m o l e c u l a r i n t e r a c t i o n s d u r i n g chromatography (10*13.)· Hydrophobic i n t e r a c t i o n chromatography (11) and l e c t i n a f f i n i t y chromatography (8) are e f f e c t i v e f o r a c r o s i n and p r o a c r o s i n p u r i f i c a t i o n , r e s p e c t i v e l y . Due t o t h e i r h i g h p i s , a c r o s i n and p r o a c r o s i n e l u t e at r e l a t i v e l y h i g h s a l t concentrations i n c a t i o n exchange chromatography, e n a b l i n g p u r i f i c a t i o n a t most pH v a l u e s u s i n g t h i s method (8,11,13.). F i n a l l y , chromatography on a f f i n i t y columns c o n j u g a t e d w i t h the i n h i b i t o r y l i g a n d p-aminobenzamidine i s an e f f e c t i v e p u r i f i c a t i o n s t e p (11,12). P r o a c r o s i n p u r i f i e d from r a b b i t t e s t e s i s 68K Mr by SDS-PAGE ( 8 ) , w h i l e t h a t p u r i f i e d from p o r c i n e (14) or o v i n e (19) spermatozoa i s 55K Mr. G u i n e a p i g t e s t i c u l a r p r o a c r o s i n i s 62E Mr, and two guinea p i g sperm p r o a c r o s i n s are 55K Mr and 43K Mr ( 1 3 ) . On the b a s i s o f these o b s e r v a t i o n s , Hardy e t a l . (13) proposed t h a t t e s t i c u l a r p r o a c r o s i n i s p r o c e s s e d , presumably p r o t e o l y t i c a l l y , t o form the lower m o l e c u l a r w e i g h t p r o a c r o s i n s found i n spermatozoa. D i r e c t evidence o f such p r o c e s s i n g has n o t y e t been r e p o r t e d . During a u t o a c t i v a t i o n of p u r i f i e d p r o a c r o s i n , m u l t i p l e a c r o s i n s d i f f e r i n g i n m o l e c u l a r weight are formed s e q u e n t i a l l y , the l a r g e s t a c r o s i n b e i n g formed f i r s t , w i t h the s m a l l e r a c r o s i n s b e i n g formed by subsequent a u t o l y s i s . A c r o s i n s generated by a u t o a c t i v a t i o n o f p u r i f i e d 55E Mr p o r c i n e sperm p r o a c r o s i n are 49K Mr ( d e s i g n a t e d m a c r o s i n ) , 38E Mr ( d e s i g n a t e d m - a c r o s i n ) , and 25E Mr ( d e s i g n a t e d m a c r o s i n ) by SDS-PAGE (14)· m ^Acrosin i s present only t r a n s i e n t l y ! b e i n g q u i c k l y degraded t o m - a c r o s i n , w h i c h i s r e l a t i v e l y more s t a b l e ( 1 4 ) . m - A c r o s i n i s formed o n l y a f t e r p r o l o n g e d i n c u b a t i o n a t b a s i c pH ( 1 4 ) . A c r o s i n s p u r i f i e d d i r e c t l y from e x t r a c t s of p o r c i n e or c a p r i n e spermatozoa are 38K Mr (11) and 42E Mr (15), r e s p e c t i v e l y , by SDS-PAGE. The s t r u c t u r a l r e l a t i o n s h i p between a c r o s i n generated from impure p r o a c r o s i n and t h a t generated from p u r i f i e d p r o a c r o s i n has n o t been determined. Thus, i t i s unknown whether a c r o s i n s p u r i f i e d d i r e c t l y from sperm e x t r a c t s are i d e n t i c a l w i t h a c r o s i n s generated by a u t o a c t i v a t i o n of p u r i f i e d p r o a c r o s i n . P r o a c r o s i n and a c r o s i n are g l y c o s y l a t e d . R a b b i t p r o a c r o s i n and p o r c i n e a c r o s i n are 8% c a r b o h y d r a t e by weight (8,16). Porcine a c r o s i n has an N - l i n k e d o l i g o s a c c h a r i d e a t Asn3 (17). Data f o r γ



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caprine a c r o s i n are consistent w i t h the r e s u l t s f o r the porcine enzyme ( 1 5 ) . The amino a c i d c o m p o s i t i o n o f v a r i o u s a c r o s i n s i n d i c a t e a r e l a t i v e l y h i g h p r o l i n e c o n t e n t (18*12)· I n t e r e s t i n g l y , when one compares t h e c o m p o s i t i o n s o f m - and m - a c r o s i n s , t h e p e p t i d e p o r t i o n removed i s r e m a r k a b l y h i g h i n p r o l i n e c o n t e n t : 31 o f t h e fragment's 85 r e s i d u e s a r e p r o l i n e ( 1 8 ) . Since t h e l i m i t e d amino a c i d sequence d a t a a v a i l a b l e i n d i c a t e t h a t a l l o f t h e N - t e r m i n a l amino a c i d s p r e s e n t i n 55K Mr p r o a c r o s i n a r e a l s o p r e s e n t i n 42E Mr a c r o s i n (discussed below), i t i s l i k e l y that the p o r t i o n l o s t on conversion of m - t o m - a c r o s i n i s a t t h e C - t e r m i n a l end. I t i s n o t y e t known whetfier t h i s u n u s u a l fragment i s removed i n t a c t o r i n p i e c e s . A c r o s i n i s i n h i b i t e d by t h e s e r i n e a c t i v e s i t e r e a g e n t s d i i s o p r o p y l f l u o r o p h o s p h a t e , p h e n y l m e t h y l s u l f o n y l f l u o r i d e , and pnitrophenyl-p'-guanidinobenzoate (9,12,15). A c r o s i n i s a l s o i n h i b i t e d by t o s y l l y s y l c h l o r o m e t h y l k e t o n e and t o s y l a r g i n y l c h l o r o methyIketone b u t n o t by t o s y l p h e n y l a l a n y l c h l o r o m e t h y l k e t o n e (£*12,15), i n d i c a t i n g s p e c i f i c i t y f o r h y d r o l y s i s on the c a r b o x y l s i d e o f l y s i n e and a r g i n i n e , and p r o v i d i n g evidence f o r an a c t i v e s i t e h i s t i d i n e . The c a t a l y t i c t r i a d o f s e r i n e p r o t e a s e a c t i v e s i t e amino a c i d residues i s present (22). I n i t i a l attempts t o determine t h e N - t e r m i n a l amino a c i d sequence o f p o r c i n e a c r o s i n were c o m p l i c a t e d by the presence o f a secondary sequence t h a t was removable a f t e r t r e a t m e n t w i t h r e d u c i n g agent i n the presence o f 6 M guanidine-HCL (20). F u r t h e r study showed t h a t t h e enzyme i s comprised o f two p o l y p e p t i d e c h a i n s l i n k e d by d i s u l f i d e bonds (12)· A s i m i l a r t w o - c h a i n s t r u c t u r e was s u b s e q u e n t l y demonstrated f o r c a p r i n e a c r o s i n ( 1 5 ) . The two c h a i n s , d e s i g n a t e d heavy and l i g h t , a r e 37K Mr and 4200 Mr, r e s p e c t i v e l y , f o r p o r c i n e a c r o s i n (12)* and 38E Mr and 3700 Mr, r e s p e c t i v e l y , f o r c a p r i n e a c r o s i n ( 1 5 ) . The s i m i l a r i t y between the amino a c i d sequences o f p o r c i n e a c r o s i n l i g h t c h a i n and t h e a c t i v a t i o n p e p t i d e s of o t h e r s e r i n e p r o t e a s e s l e d Fock-Nuzel e t a l . (17) t o propose t h a t the mechanism o f p r o a c r o s i n a c t i v a t i o n i s l i m i t e d h y d r o l y s i s o f t h e s i n g l e p r o a c r o s i n p o l y p e p t i d e c h a i n t o produce the t w o - c h a i n a c t i v e enzyme. Subsequent sequencing o f p o r c i n e p r o a c r o s i n showed t h a t i t s N - t e r m i n a l sequence i s i d e n t i c a l t o t h a t o f p o r c i n e a c r o s i n l i g h t c h a i n , and t h e N - t e r m i n a l sequence o f t h e heavy c h a i n i s encountered i n the p r o a c r o s i n sequence i m m e d i a t e l y a f t e r the C - t e r m i n a l a r g i n i n e of the l i g h t c h a i n (A. Henschen, p e r s o n a l communication), thus p r o v i d i n g f u r t h e r s u p p o r t f o r t h e proposed a c t i v a t i o n mechanism. D u r i n g i n i t i a l a u t o a c t i v a t i o n o f guinea p i g p r o a c r o s i n s , a 5000 + 1000 Mr fragment ( l i g h t c h a i n ) i s generated which remains bound t o the remainder o f t h e a c r o s i n molecule (heavy c h a i n ) b y d i s u l f i d e bonds, r e g a r d l e s s o f whether t h e p r o a c r o s i n i s the 62K Mr s i z e v a r i a n t i s o l a t e d from t e s t i s o r t h e 55K Mr o r 43K Mr s i z e v a r i a n t s i s o l a t e d from spermatozoa (15.). These l a t t e r r e s u l t s a r e p r e s e n t l y the o n l y d i r e c t evidence f o r a p r o a c r o s i n a c t i v a t i o n mechanism i n v o l v i n g l i m i t e d p r o t e o l y s i s of a single polypeptide chain t o produce a t w o - c h a i n a c r o s i n . The sequences o f p o r c i n e and c a p r i n e a c r o s i n l i g h t c h a i n s and the N - t e r m i n a l sequences o f t h e a c r o s i n heavy c h a i n s a r e shown a l i g n e d w i t h t h e sequences o f s e v e r a l s e r i n e p r o t e a s e s i n F i g u r e 1. Sequence s i m i l a r i t y between p o r c i n e and c a p r i n e a c r o s i n s i s r e a d i l y apparent, as i s t h e s i m i l a r i t y o f t h e a c r o s i n sequences (range o f



1 10 20 30 40 50 WGGMSAEPG AWPWMVSLQI PMYHNNRRYH TCGGILLNSH WVLTAAHCFK NK IIGGODAAHG SWPWMVSLOI FTYHNNRRYH VCGGSLL WGGCVAHPH SWPWOVSLRT RFG MH FCGGTLISPE WVLTAAHCL- -IVGGRDTSLG RWPWQVSL-RYDGAH LCGGSLLSGD WVLTAAHCFIVNGEEAVPG SWPWOVSLWD KTG FH FCGGSLINEN WWTAAHCGWGGTEAQRN SWPSQISLQY RSG--SSWAH TCGGTLIRON WVMTAAHCVD WGGTRAAQG EFPFMVRL- - SMG CGGALYAOD IVLTAAHCVS IVGGYTCGAN TVPYOVSLN- -SG YH FCGGSLINSQ WWSAAHCYIVEGQDAEVG LSPWQVMLF- - -RKSPOE-L LCGASLISDR WVLTAAHCLL YP IIGGRESRPH SRPYMAYLOI ---OSPAGOS RCGGFLVRED FVLTAAHCWIKGGLFADIA SHPWQAAIFA KHRRSPGERF LCGGILISSC WILSAAHCF- -NIHG RGGGALLGDR WILTAAHTL- YP IIGGQKAKMG NFPWQVFT-IVGG A G S PWQVSL SG H FCGG LI WVLTAAHC G Ρ CG V AAHC

Acrosin heavy chain (po) Acrosin heavy chain (ca) Plasmin (hu) Hepsin (hu) Chymotrypsin (bo) Elastase (po) TrvDsin (S. griseus) Trypsin (bo) Prothrombin (bo) Cathepsin G (hu) Plasminogen activator (hu) C l r b-chain (hu) Conserved Invariable

70% 48% 48% 46% 44% 42% 40% 38% 35% 35% 33% 77% 100%

Positional identity

Reference 20 15 68 24 25 29 28 27 30 32 26 31 33 33

Figure 1. Sequences of acrosin l i g h t chains and N-terminal sequences of acrosin heavy chains shown aligned with the sequences of corresponding portions of other serine proteases. Positions i d e n t i c a l with the porcine acrosin sequence are underlined. Alignments for plasmin, prothrombin, C l r b-chain, cathepsin G, and plasminogen activator are from (24), except minor adjustments were made to maximize s i m i l a r i t y with acrosin. Sequence s i m i l a r i t i e s expressed as percent p o s i t i o n a l i d e n t i t y to the porcine sequences are indicated to the right of each sequence. Abbreviations: po - porcine, ca - caprine, bo - bovine, hu - human.

78% 40% 26% 22% 17% 0%

Acrosin l i g h t chain (po) Acrosin l i g h t chain (ca) Chymotrypsinogen (bo) Heps i n (hu) Plasminogen activator (hu) Plasminogen (hu) Trypsinogen (bo)

Reference 17 15 25 24 26 68 27

1 10 20 RDNATCDGPC GLRFRQKLES GM-R RDNTTCDGPC GIRFRONR Ç GVPAIQPVLS GLSR RFLAAICQDC GRRKLP -VDR YCDVPSCSTÇ GLROYSQPOF — R PQCAAPSFDÇ GKPQMEPKKC PG-R VD DDDK

Positional identity


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positional i d e n t i t i e s , 33% to 48%) with those of the other serine proteases shown. A majority (77%) of the amino acids that are conserved in serine proteases, and a l l of those that are invariable, are also conserved in the p a r t i a l sequences of acrosin available (20,22K thus, acrosin is homologous (21) with the trypsin family of serine proteases. Similar comparisons using a longer acrosin sequence (124 residues) produced similar positional i d e n t i t i e s , leading Fock-Nutzel (22) to the remarkable proposal that acrosin diverged from the other serine proteases a f t e r Streptomyces griseus trypsin but before pancreatic trypsin and thrombin during the evolution of the serine proteases. Using the limited sequence comparisons shown i n Figure 1 and a divergence time for the porcine and caprine lineages of SO m i l l i o n years ago. Hardy et a l . (15) calculated a unit evolutionary period (time required for a 1% change in amino acid sequence) of 1.8 m i l l i o n years for acrosin. This sequence divergence rate i s more than three times as rapid as that of trypsin, and similar to those of enzymes such as carbonic anhydrase, ribonuclease, and lactalbumin (23). The portions of the acrosin sequence compared for this c a l c u l a t i o n are homologous with the other serine proteases. Since the C-terminal portion of m -acrosin, and therefore proacrosin necessarily, has an unusually high proline content, i t seems u n l i k e l y that this region of the acrosin sequence is homologous to the other serine proteases. It w i l l be very interesting to compare the amino acid sequence divergence rate of this portion of the acrosin molecule, as i t might be expected to be involved i n functions unique to acrosin. THE FUNCTIONAL DOMAINS OF ACROSIN PROTEOLYTIC ACTIVITY. In comparison with pancreatic trypsin, acrosin is 50% larger, contains carbohydrate, and is more hydrophobic (11). Although acrosin has often been referred to as t r y p s i n - l i k e , the above structural differences are reflected i n k i n e t i c and hydrolytic s p e c i f i c i t y differences, in d i f f e r e n t interactions with membranes, and in the newly described l e c t i n function of acrosin. Substrate s p e c i f i c i t y differences between boar acrosin and trypsin are not p a r t i c u l a r l y manifest when using small substrates, but these enzymes show d i s t i n c t l y d i f f e r e n t k i n e t i c s of porcine ZP hydrolysis (34). The loss of 30% mass in the conversion from m - to m -acrosin has l i t t l e effect on the k i n e t i c analyses of i n h i b i t i o n and substrate preference with a r t i f i c i a l substrates and small trypsin i n h i b i t o r s , indicating that this excised portion of the enzyme contributes l i t t l e to the topography of the active site (.35). From Κηι analyses with amide and ester substrates of Arg and Lys, acrSIin prefers the Arg substrates over Lys, and Km differences between amide and ester substrates indicates that acSSsin proceeds k i n e t i c a l l y through a c l a s s i c a l double displacement mechanism as does trypsin (36). Acrosin causes the limited and s p e c i f i c cleavage of only certain portions of the ZP (7,32,38)· The ZP is composed of three families of glycoproteins, denoted 9OK, 55K and 55K , and two derived components, 65K and 25K (39). The f>5K and 25K components are produced from the 90K family by a p r o t e o l y t i c event in the f o l l i c l e . The 90K family, and the 90K and 65K components, were s p e c i f i c a l l y &


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hydrolyzed by acrosin to small polypeptides not retained in SDS-PAGE (40). The 55K families were not affected by acrosin in the particulate or s o l i d forms of the ZP. The ZP remained v i s u a l l y intact even after extended acrosin hydrolysis reduced the 9OK family to small polypeptides (40), and the interacting hydrolyzed peptides could only be liberated with mercaptoethanol and dénaturants. ZONA PELLUCIDA BINDING. Several molecular mechanisms have been proposed for sperm-egg binding. Sperm binding to the ZP has been suggested to involve carbohydrate-protein interaction, proteinprotein interaction, or both (41). I n h i b i t i o n of mouse sperm binding to mouse eggs was observed with trypsin i n h i b i t o r s (42,43), and a n t i acrosin antibodies inhibited sperm binding in the rabbit (44). Two molecules that have trypsin binding s p e c i f i c i t y were observed on mouse sperm: a molecule that turned over a c t i v e - s i t e t i t r a n t inhibitors of trypsin, but that was not i t s e l f c a t a l y t i c a l l y active (46)« and a molecule that bound seminal plasma i n h i b i t o r (45). C a t a l y t i c a l l y active acrosin bound ZP p r e f e r e n t i a l l y over a control protein, as would be expected from a normal enzyme-substrate relationship (34). A possible explanation of the above data i s that the p r o t e o l y t i c substrate binding s i t e of acrosin, or a portion of the molecule that retains binding a c t i v i t y after autolysis, resides on the sperm surface and i s involved in binding ZP. When a l l acrosins (including small a u t o l y t i j ^ o r m s of 12-18K Mr) were Western blotted, a l l of the forms bound I-ZP even when the enzyme was inhibited with DFP (47-49). This implied a second binding site on acrosin for ZP. Recently, a fucose binding protein with a molecular weight corresponding to boar proacrosin, 53K, and having the same N-terminal sequence as acrosin (50), was described on the surface of acrosome intact boar sperm (51). The carbohydrates that bound acrosin inhibited sperm binding (52), and these carbohydrates also inhibited the amidase a c t i v i t y of acrosin (53). In addition to acrosin, low molecular weight polypeptides involved i n the species-specific binding of rabbit sperm to rabbit ZP were described as fucose binding l e c t i n s (54). These data are consistent with acrosin binding a fucose containing ligand on the ZP, and also suggest that the binding site for fucose resides in the small Mr forms of acrosin. The i n h i b i t i o n of acrosin by polysulfated polysaccharides is interesting since d i f f e r e n t acrosins are inhibited d i f f e r e n t i a l l y . The m - and subsequent hydrolytic products of m -acrosin are not as susceptible to i n h i b i t i o n with these carbohydrates as is m -acrosin (53). These data provide evidence for a model for the penetration of ZP by sperm by a sequential binding, hydrolysis, and subsequent rebinding that would be necessary for sperm penetration of the ZP. HYDROPHOBIC BINDING. Acrosin is an unusual protease in that i t has high a f f i n i t y for l i p i d s and detergents, and i t s amidolytic a c t i v i t y is modified by these agents. Boar acrosin aggregates with i t s e l f and with other proteins in the absence of detergents (10,55). Its hydrophobic nature (11) and l i p o p h i l i c properties led to i t s description as an e x t r i n s i c membrane protein. Non-ionic detergents stimulated acrosin's amidase a c t i v i t y at c r i t i c a l micelle concentrations of those detergents (56), but the enzyme did not bind to a micelle of T r i t o n X-100 (56).



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m - A c r o s i n has been r e p o r t e d t o be i n h i b i t e d b y p h o s p h o l i p i d v e s i c l e ! ( 5 6 ) , whereas a n i o n i c p h o s p h o l i p i d d i s p e r s i o n s s t i m u l a t e d the a u t o l y s i s o f p r o a c r o s i n ( 5 7 ) . W i t h d e t e r g e n t couple e l e c t r o p h o r e s i s ( 5 8 ) , where a m p h i p h i l i c p r o t e i n s b i n d charged d e t e r g e n t couples r e s u l t i n g i n a m o b i l i t y s h i f t and h y d r o p h i l i c p r o t e i n s a r e u n a f f e c t e d , p r o a c r o s i n , m - and m - a c r o s i n , p e p t i d e s r e l e a s e d from a c r o s i n a u t o l y s i s , and a ?8K b i n d i n g p r o t e i n , behaved as a m p h i p h i l i c o r i n t r i n s i c membrane p r o t e i n s (Urch, u n p u b l i s h e d o b s e r v a t i o n s ) . Since the 28K-proacrosin i n t e r a c t i o n r e q u i r e d e i t h e r 8 M urea or 6 M guanidine HC1 t o d i s r u p t i t ( 1 0 ) , t h i s p r o t e i n - p r o t e i n i n t e r a c t i o n may a c t t o anchor p r o a c r o s i n t o the acrosomal membranes or t o o t h e r p r o a c r o s i n m o l e c u l e s . W h i l e a c r o s i n does n o t f i t a l l t h e c r i t e r i a f o r an i n t r i n s i c membrane p r o t e i n , a c r o s i n was a s s o c i a t e d w i t h and e x t r a c t e d from p r e p a r a t i o n s o f sperm membrane v e s i c l e s w i t h 1 mM HC1 and T r i t o n X-100 (59, U r c h , u n p u b l i s h e d o b s e r v a t i o n s ) . Salt o r c o m b i n a t i o n s o f s a l t and d e t e r g e n t were i n e f f e c t i v e . The i n t e r a c t i o n o f a c r o s i n w i t h membranes, p a r t i c u l a r l y sperm acrosomal membranes, i s o b v i o u s l y important i n ZP b i n d i n g and p e n e t r a t i o n . Thus, w h i l e the exact d e f i n i t i o n o f a c r o s i n ' s i n t e r a c t i o n w i t h membranes remains t o be e x p l a i n e d , i t i s apparent t h a t a c r o s i n h y d r o p h o b i c i t y i s important t o i t s f u n c t i o n i n f e r t i l i z a t i o n . THE CELLULAR FUNCTION OF ACROSIN AS A SPERM LYSIN. A c r o s i n f u l f i l l s most o f the above l i s t e d c r i t e r i a f o r a sperm l y s i n . I t has been l o c a l i z e d onto b o t h t h e i n n e r and o u t e r acrosomal membranes by h i s t o c h e m i c a l and immunochemical l o c a l i z a t i o n procedures (60,61), and i s a t t h e c o r r e c t p l a c e t o f u n c t i o n i n f e r t i l i z a t i o n . Complete ZP d i s s o l u t i o n by homologous a c r o s i n s , t h e second l y s i n c r i t e r i o n , i s not observed w i t h a l l s p e c i e s . W i t h b o a r a c r o s i n , d i s s o l u t i o n o f t h e p o r c i n e ZP was never v i s u a l l y observed, as was t r u e w i t h sheep ZP and ram a c r o s i n ( 6 2 ) . To v i s u a l i z e t h e e f f e c t o f a c r o s i n on t h e ZP, e l e c t r o p h o r e t i c o r o t h e r s e p a r a t i o n p r o c e d u r e s t h a t employed dénaturants and d i s u l f i d e bond r e d u c t i o n were r e q u i r e d (7,37,38). Complete d i s s o l u t i o n o f t h e ZP does n o t occur i n normal f e r t i l i z a t i o n , s i n c e the ZP i s needed f o r the c o n t i n u e d development o f t h e embryo. The t h i r d c r i t e r i o n o f t h e i n h i b i t i o n o f f e r t i l i z a t i o n by a c t i v e s i t e i n h i b i t o r s was observed b o t h i n v i t r o (63) and i n v i v o (64) f e r t i l i z a t i o n . However, t h i s i n h i b i t i o n was never t o t a l , and once t h e sperm had t i g h t l y bound t h e ZP, t r y p s i n a c t i v e s i t e i n h i b i t o r s d i d not prevent f u r t h e r p e n e t r a t i o n o r h y d r o l y s i s o f the ZP ( 6 5 ) . T h e r e f o r e , w h i l e a c r o s i n does n o t s t r i c t l y f o l l o w a l l o f t h e c r i t e r i a f o r a ZP l y s i n , i t does cause l i m i t e d h y d r o l y s i s o f t h e ZP and i t i s i n the c o r r e c t p l a c e t o a c t i n p e n e t r a t i o n o f t h e ZP. W h i l e a c r o s i n has been l o c a l i z e d t o t h e acrosome, t h e r e a r e c o n f l i c t i n g r e p o r t s as t o e x a c t l y where i n the acrosomal compartment a c r o s i n i s l o c a l i z e d b e f o r e t h e acrosome r e a c t i o n and t h e t i m i n g o f i t s r e l e a s e i n the f e r t i l i z a t i o n p r o c e s s . From the d e s c r i p t i o n o f a c r o s i n as a ZP b i n d i n g l e c t i n , a c r o s i n must be a v a i l a b l e t o b i n d t h e ZP, and t h i s would p l a c e the m o l e c u l e on t h e s u r f a c e o f the i n t a c t sperm, o r i n some s p e c i e s , o f t h e aerosome-reacted sperm. I n t h e mouse, o n l y acrosome i n t a c t sperm b i n d t h e ZP ( 6 6 ) . Once t h e sperm has undergone t h e acrosome r e a c t i o n and t h e o u t e r membranes ( l i m i t i n g


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plasma membrane and o u t e r acrosomal membrane) have v e s i c u l a t e d , the i n n e r acrosomal membrane becomes the l i m i t i n g s u r f a c e o f the sperm and o f importance i n gamete i n t e r a c t i o n s . I n the hamster, acrosome r e a c t e d sperm b i n d t o the ZP, and the f e r t i l i z i n g sperm appears t o remain bound t o the ZP by a remnant o f the v e s i c u l a t e d acrosomal membrane o r acrosomal ghost (67.). W i t h boar sperm, a c r o s i n has been l o c a l i z e d t o the acrosomal s u r f a c e o f l i v e sperm w i t h immunofluorescent t e c h n i q u e s ( U r c h , u n p u b l i s h e d o b s e r v a t i o n s ) . From these o b s e r v a t i o n s , a c r o s i n i s i n the c o r r e c t p l a c e t o i n t e r a c t w i t h the ZP.


STRUCTURE-FUNCTION MODELS OF ACROSIN Any attempt a t p r o p o s i n g a model f o r a c r o s i n ' s s t r u c t u r e - f u n c t i o n r e l a t i o n s must n e c e s s a r i l y generate a w o r k i n g h y p o t h e s i s , due t o i n s u f f i c i e n t d a t a and the m u l t i p l e and dynamic m o l e c u l a r forms o f a c r o s i n t h a t e x i s t . A c r o s i n i s much l i k e the Greek god o f dreams, Phantasos, i n t h a t i t changes i t s forms v i a a u t o l y s i s so q u i c k l y and r e a d i l y t h a t i t seems almost p h a n t o m - l i k e . T h i s polymorphism, and presumably the m u l t i p l e pathways l e a d i n g t o these m u l t i p l e forms, p r o v i d e s a g r e a t e x p e r i m e n t a l c h a l l e n g e t o those o f us who w i s h t o u n d e r s t a n d the s t r u c t u r e - f u n c t i o n r e l a t i o n s o f a c r o s i n . PROCESSING OF ACROSIN'S FUNCTIONAL DOMAINS. I n F i g u r e 2, the processing v i a a u t o l y s i s of a c r o s i n i s i l l u s t r a t e d beginning w i t h p r o a c r o s i n . F i g u r e 2 i s h i g h l y schematic and does n o t i n c l u d e a l l of the s t e p s i n a c r o s i n p r o c e s s i n g o r a l l o f i t s m o l e c u l a r forms. The purpose o f the scheme i s t o p r o v i d e a r e a s o n a b l e s u g g e s t i o n as t o the r e l a t i o n o f a c r o s i n ' s f u n c t i o n a l domains and how these domains might be h y d r o l y t i c a l l y p r o c e s s e d t o generate m o l e c u l e s which have d i f f e r e n t c a t a l y t i c and b i n d i n g p r o p e r t i e s . The s i n g l e g l y c o s y l a t e d p o l y p e p t i d e c h a i n p r o a c r o s i n i s c o n v e r t e d from an i n a c t i v e form t o an a c t i v e form by l i m i t e d p r o t e o l y s i s i n the N - t e r m i n a l r e g i o n o f the p o l y p e p t i d e c h a i n . I n the case o f boar sperm a c r o s i n , t h i s p r o c e s s i n g o c c u r s between Arg22 and V a l 2 3 t h e r e b y p r o d u c i n g a twoc h a i n e d m o l e c u l e . The l i g h t (L) and heavy (H) c h a i n s a r e c r o s s l i n k e d v i a d i s u l f i d e bonds. A d d i t i o n a l p r o c e s s i n g o c c u r s i n the C - t e r m i n a l p o r t i o n o f the H c h a i n e v e n t u a l l y p r o d u c i n g an e n z y m a t i c a l l y a c t i v e m - a c r o s i n . These two p r o a c r o s i n p r o c e s s i n g events p r o b a b l y o c c u r by more than one pathway and produce s e v e r a l i n t e r m e d i a t e forms. The p r o t e o l y t i c a l l y f u n c t i o n a l molecule i s a d d i t i o n a l l y p r o c e s s e d such t h a t the t h r e e f u n c t i o n a l domains o f the enzyme are s e p a r a t e d . Two pathways are suggested, one which s e p a r a t e s the c a r b o h y d r a t e b i n d i n g domain from the hydrophobic and p r o t e o l y t i c domains w h i l e the o t h e r pathway s e p a r a t e s the p r o t e o l y t i c domain from the hydrophobic and c a r b o h y d r a t e b i n d i n g domains. Thus, membrane bound a c r o s i n forms a r e produced which can h y d r o l y z e p e p t i d e bonds o r b i n d t o c a r b o h y d r a t e r e s i d u e s (m - f o r m s ) . F u r t h e r p r o c e s s i n g s e p a r a t e s a l l domains from each o t h e r p r o d u c i n g a m i x t u r e o f m o l e c u l e s w i t h Mr's o f 12-18K w h i c h i n d i v i d u a l l y poss ess hydrophobic b i n d i n g and c a r b o h y d r a t e b i n d i n g domains and a n o n - f u n c t i o n a l p r o t e o l y t i c domain. Presumably, c o m p l e t e l y p r o c e s s e d a c r o s i n m o l e c u l e s (12-18K) w i t h s e p a r a t e d domains would no l o n g e r be f u n c t i o n a l l y i m p o r t a n t i n the sperm binding/penetration process.




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all 12 - 1ΘΚ F i g u r e 2. P r o c e s s i n g o f A c r o s i n ' s F u n c t i o n a l D o m a i n s . s y m b o l s u s e d a r e d e f i n e d i n t h e l e g e n d o f F i g u r e 3.

The



15.

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Structure-Function Properties of Acrosin

AN ACROSIN MECHANISM FOR ZP BINDING AND PENETRATION BY SPERM. F i g u r e 3 i s a scheme i l l u s t r a t i n g the way m - a c r o s i n may a s s i s t the sperm t o b i n d and p e n e t r a t e the ZP. A c r o s i n i s bound t o the sperm membrane v i a i t s hydrophobic domain. The scheme suggests t h a t a c r o s i n may be an i n t r i n s i c membrane g l y c o p r o t e i n i n t h a t the h y d r o p h o b i c domain i s i n s e r t e d i n t o t h e sperm membrane. However, a l t e r n a t e schemes a r e p o s s i b l e i n c l u d i n g an e x t r i n s i c r a t h e r t h a n i n t r i n s i c mode o f b i n d i n g o r b i n d i n g t o the sperm membrane v i a an i n t e r m e d i a t e " a c r o s i n b i n d i n g p r o t e i n " ( 1 0 ) . B i n d i n g o f the sperm t o the ZP i s v i a the carbohyd r a t e b i n d i n g s i t e . While t h e a c r o s i n i s bound t o the ZP, i t s p r o t e o l y t i c s i t e a c t i v i t y i s reduced. Thus, when a c r o s i n i s f u n c t i o n i n g i n t h e b i n d i n g mode i t may n o t be f u l l y f u n c t i o n a l i n i t s p r o t e o l y t i c mode. Other a c r o s i n m o l e c u l e s on the sperm membrane would i n t e r a c t w i t h the ZP v i a the p r o t e o l y t i c s i t e , t h e r e b y h y d r o l y z i n g p e p t i d e bonds and a l l o w i n g the m o t i l e sperm t o p r o g r e s s i v e l y p e n e t r a t e t h e ZP m a t r i x . P o s s e s s i o n o f b o t h b i n d i n g and h y d r o l y z i n g domains would p e r m i t a c r o s i n t o h y d r o l y z e a p e n e t r a t i o n p a t h w h i l e s t i l l b i n d i n g the m o t i l e sperm t o the ZP. R e l e a s e from the Z P - a c r o s i n b i n d i n g mode t o p e r m i t p r o g r e s s i v e sperm p e n e t r a t i o n c o u l d be v i a t h r e e mechanisms: a) t h e c a r b o h y d r a t e b i n d i n g domain-ZP i n t e r a c t i o n i s dynamic and c o u l d s i m p l y be c h e m i c a l l y r e v e r s e d , b) t h e a c r o s i n c a r b o h y d r a t e b i n d i n g domain c o u l d be p r o c e s s e d ( h y d r o l y z e d ) by another a c r o s i n m o l e c u l e , and c) the bound ZP o l i g o s a c c h a r i d e c o u l d be h y d r o l y z e d from t h e ZP g l y c o p r o t e i n by an a c r o s i n m o l e c u l e and the l i g a n d - o l i g o s a c c h a r i d e r e l e a s e d as a glycopeptide. Mechanism " a " i n v o l v e s a c y c l i n g o f b i n d i n g - r e l e a s e s t e p s between a c r o s i n and the ZP. Mechanism "b" i s a n o n - c y c l i n g p r o c e s s where the c a r b o h y d r a t e b i n d i n g f u n c t i o n i s a s i n g u l a r e v e n t . Mechanism " c " i s the l e a s t l i k e l y o f the t h r e e mechanisms p o s t u l a t e d as p r o t e o l y s i s by b o a r a c r o s i n i s l i m i t e d t o one (90E) o f t h e t h r e e g l y c o p r o t e i n f a m i l i e s o f the p i g egg ZP. One o r b o t h o f the o t h e r ZP components (55K and 55K ) f u n c t i o n as l i g a n d s f o r sperm b i n d i n g . Thus, u s i n g the l e c t i n - p r o t e a s e f u n c t i o n s o f a c r o s i n , t h r o u g h a s e r i e s o f p r o t e o l y t i c and c y c l i n g / n o n - c y c l i n g c a r b o h y d r a t e b i n d i n g e v e n t s , a c r o s i n c o u l d p r o v i d e the n e c e s s a r y c e l l u l a r b i n d i n g and p e n e t r a t i o n f u n c t i o n s f o r t h e m o t i l e sperm t o t r a v e r s e t h e ZP on i t s way t o f u s i o n w i t h the egg plasma membrane, the subsequent s t e p i n the f e r t i l i z a t i o n p r o c e s s . FUTURE RESEARCH A p r i m a r y focus o f f u t u r e r e s e a r c h s h o u l d be d e t e r m i n a t i o n o f t h e p r i m a r y (10) s t r u c t u r e o f a c r o s i n . P r o t e i n sequencing by the c l a s s i c a l Edman d e g r a d a t i o n method and recombinant DNA methods w i l l be needed t o e l u c i d a t e c o m p l e t e l y the p r i m a r y s t r u c t u r e o f a c r o s i n . Knowledge o f the p r i m a r y s t r u c t u r e i s o f fundamental importance f o r a d d i t i o n a l s t u d i e s on the f u n c t i o n a l p r o p e r t i e s o f the enzyme. W i t h the 1 s t r u c t u r e known, t h e f u n c t i o n a l domains o f the enzyme can be more r e a d i l y d e f i n e d . By comparison w i t h the p r o t e o l y t i c s i t e o f o t h e r s e r i n e a c t i v e s i t e p r o t e a s e s , i t s h o u l d be p o s s i b l e t o deduce b i n d i n g and c a t a l y s i s f u n c t i o n s t h a t a r e common t o s e r i n e p r o t e a s e s where s t r u c t u r a l homology i s h i g h , and t o pursue the d i s c o v e r y o f unique f u n c t i o n s where homology i s low. The s t r u c t u r e o f the c a r b o h y d r a t e b i n d i n g domain can be compared w i t h o t h e r fucose


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PM

ZP

F i g u r e 3. A Model f o r A c r o s i n ' s R o l e i n Zona P e l l u c i d a B i n d i n g and P e n e t r a t i o n o f Sperm. The model uses the m ^ - a c r o s i n form d e p i c t e d i n F i g u r e 2. A c r o s i n i s bound to the sperm plasma membrane (PM) v i a i t s h y d r o p h o b i c domain and i n t e r a c t s w i t h the egg zona p e l l u c i d a (ZP) v i a i t s p r o t e a s e and c a r b o h y d r a t e b i n d i n g domains. The ZP i s composed o f t h r e e g l y c o p r o t e i n s which have u n i q u e p o l y p e p t i d e c h a i n s and b o t h s h a r e d and u n i q u e o l i g o saccharide moieties. Symbols u s e d : 1 , · , ^ — o l i g o s a c c h a r i d e m o i e t i e s , s h a r e d and u n i q u e ; = f u n c t i o n a l domain; Q - non f u n c t i o n a l domain; Ρ = protease active s i t e ; Η « hydrophobic binding site; C - carbohydrate b i n d i n g s i t e ; \ - d i s u l f i d e bond.



15. HEDRICK ET AL.


in

l e c t i n s , a g a i n s e a r c h i n g f o r homology/analogy and h e t e r o l o g y / ^ uniqueness i n u n d e r s t a n d i n g i t s f u n c t i o n . Knowledge o f the 1 s t r u c t u r e can a s s i s t i n u n d e r s t a n d i n g the m o l e c u l a r n a t u r e o f a c r o s i n b i n d i n g t o membranes and perhaps a l s o o r g a n i z a t i o n o f p r o a c r o s i n i n the acrosomal m a t r i x . U n d e r s t a n d i n g the a c t i v a t i o n p r o c e s s ( p r o a c r o s i n t o a c r o s i n ) r e q u i r e s a knowledge o f the 1° s t r u c t u r e . R e g u l a t i o n o f a c r o s i n a c t i v i t y a l s o i n c l u d e s l i m i t e d p r o t e o l y s i s ( a u t o l y s i s ) which modulates and s e p a r a t e s the f u n c t i o n a l domains o f the enzyme from one a n o t h e r , so t h a t we can determine what forms o f the enzyme are "where and when"; i n f o r m a t i o n e s s e n t i a l f o r u n d e r s t a n d i n g the c e l l u l a r function of acrosin. Knowledge o f a c r o s i n ' s s t r u c t u r e w i l l f a c i l i t a t e the i d e n t i f i c a t i o n o f the s u b s t r a t e ( s ) and l i g a n d ( s ) s i t e s o f the ZP g l y c o p r o t e i n s . The ZP g l y c o p r o t e i n s are complex and d i f f i c u l t t o study. P r o g r e s s toward u n d e r s t a n d i n g the r o l e o f a c r o s i n ' s " p a r t n e r m o l e c u l e s " i n sperm b i n d i n g and p e n e t r a t i o n o f the ZP w i l l be g r e a t l y a s s i s t e d by a d d i t i o n a l understanding o f acrosin's s t r u c t u r e . From the u n d e r s t a n d i n g gained i n the above s t u d i e s w i l l come the p o t e n t i a l t o c r e a t e s p e c i f i c chemical r e a g e n t s which can a l t e r / i n h i b i t a c r o s i n ' s m o l e c u l a r i n t e r a c t i o n s and the b i n d i n g / p e n e t r a t i o n of the ZP. T h i s w i l l p r o v i d e the needed u n e q u i v o c a l e v i d e n c e o f a c r o s i n ' s r o l e i n f e r t i l i z a t i o n and a b a s i c u n d e r s t a n d i n g o f gamete i n t e r a c t i o n s , knowledge which i s p o t e n t i a l l y u s e f u l f o r r e g u l a t i n g the f e r t i l i z a t i o n p r o c e s s . ACKNOWLEDGMENTS T h i s work was supported i n p a r t b y USPHS HD04906 ( J L H ) , HD07088 (DMH), and USDA 85-CRCR-1-1855 (UAU and J L H ) . We thank N.J. W a r d r i p f o r a s s i s t a n c e w i t h the f i g u r e s . LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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