abstract fertilization is one of the most specific and carefully regulated cell

7/17/2019 Abstract Fertilization is One of the Most Specific and Carefully Regulated Cell

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Abstract Fertilization is one of the most specic and carefully regulated cell-cell

interactions in the animal body and is determined to a large extent by

compatibility between ligand and receptor molecules on the surface of each

gamete. On the zona pellucida (Z!" sperm receptor acti#ity is associated with

glycoproteins Z$ (primary receptor for acrosome-intact sperm! and Z%

(secondary receptor for acrosome-reacted sperm! but their complementarybinding proteins on sperm are less well dened. &n this communication we

re#iew the e#idence for proacrosin as a secondary Z binding protein.

roacrosin'acrosin binds non-enzymically to Z glycoproteins. inding is a

strong ionic interaction between polysulphate groups on Z glycoproteins

(probably on their carbohydrate moieties! and basic residues on the surface of

proacrosin. )he stereochemistry of the reactants is crucial and determines to a

large extent the a*nity of binding. +ite-directed mutagenesis and a $,-

structural analysis of boar and ram acrosin ha#e identied % clusters of basic

residues potentially in#ol#ed in binding. A polysulphonated anticancer drug"

suramin" has been shown to bind strongly to proacrosin'acrosin and to inhibit

spermegg binding in #itro. &n the mouse model" %/&-Z% and $ 0-suramin

bind 1/2 less e3ecti#ely to acrosin 4null5 sperm than to wild-type sperm.

6either Z% nor suramin bind to acrosome intact sperm and can" therefore"

only exert their e3ects after exposure of the acrosomal contents. O#erall" this

combination of biochemical" genetic and functional data supports the

hypothesis that proacrosin is a multifunctional protein with a signicant role in

retaining acrosome-reacted sperm on the Z surface long enough to enable Z

penetration to begin. 7 %88% 9lse#ier +cience &reland :td. All rights reser#ed.

. &ntroduction ,uring their passage from the testis to the site of

fertilization in the o#iduct" mammalian spermatozoa encounter a #ariety

of di3erent cell-types ranging from +ertoli cells and spermatozoa in the

seminiferous tubules to epithelial cells in the epididymis" uterus and

o#iduct and nally the cumulus oophorus. Although in many species

spermatozoa show a transient interaction with some of these cell-types

that may ha#e physiological signicance (e.g. for sur#i#al in the uterus!"

none of the cells nor their secretory products elicit the appropriate

signalling pathways that are initiated when the spermatozoon nally

reaches the egg and binds to the zona pellucida (Z!. &n part" this may

be owing to the immaturity of the spermatozoa themsel#es" thepresence of inhibitory'membrane stabilizing factors or the incorrect

combination of signals emanating from the target cell. )he specicity of

fertilization" therefore" resides to a large extent in the complementary

cross-tal; that occurs between homologous gametes. <ithin this

scenario" the ligandreceptor complexes present on the surface of sperm

and egg ob#iously play a ;ey role. As a result of a large amount of

accumulated research on the cell biology of fertilization in di3erent



species (for re#iew see =anagimachi" >>?!" mammalian spermegg

interactions can be #iewed as a series of se@uential steps. &n the mouse

paradigm (<assarman" >>>!" an acrosome-intact spermatozoon arri#ing

on the surface of the Z recognizes and binds to a primary receptor

associated with the O-lin;ed carbohydrate moiety of glycoprotein Z$

(Fig. !. )his binding is of su*cient a*nity to retain the motilespermatozoon on the surface of the Z while at the same time

permitting clustering of receptorligand complexes that induce

exocytosis of the acrosomal #esicle. 9xtensi#e fenestration and loss of

the acrosomal cap exposes the acrosomal matrix and e#entually the

inner acrosomal membrane. At this stage a second binding step ta;es

place between the secondary receptor on the Z (glycoprotein Z%! and

a binding protein within the acrosomal matrix and'or on the inner

acrosomal membrane. inding" howe#er" must not be so tenacious that

it pre#ents the sperm head from penetrating through the Z. artly as a

result of thrust forces generated by the motile agellum and partly due

to an unidentied 4lyticli;e5 action" the sperm head penetrates through

the Z and accesses the peri#itilline space (edford" >>B!. 0ere a third

binding step ensues that possibly in#ol#es integrin or C,> receptors on

the oolema (Diller et al." %888!. <hilst this picture seems fairly clear" the

nature of the primary" secondary and tertiary binding proteins on

spermatozoa is highly contentious. A wide #ariety of candidates has

been described ()able !" many of them in the

mouse system alone. )hese are su*ciently disparate in their properties to raise

important @uestions about how they are integrated with one another" e#en

allowing for redundancy within the system as suggested by recent gene4;noc;out5 experiments. )he relati#e merits of these di3erent Z binding

proteins ha#e been discussed in se#eral re#iews (Dc:es;ey" >>BE <assarman"

>>>!. &n this communication we shall focus on the e#idence in fa#our of a

sperm-specic protein called proacrosin'acrosin as a secondary binding or

ligand molecule for retaining acrosome-reacted sperm on the zona surface.

Although acrosin has long been regarded as a zona lysin" the e#idence for this

is less con#incing than pre#iously supposed (edford" >>B! and it should now

be regarded as a multifunctional protein. %. 9#idence for sperm

proacrosin'acrosin as a Z-binding protein )he initial experiments to detect Z

binding proteins on spermatozoa were based on the principle of using Z% and

Z$ receptors as probes to identify their respecti#e binding proteins in extracts

of spermatozoa. For this purpose" detergent-soluble proteins from boar sperm

were separated by +,+A9" displayed on a <estern blot and incubated with

%/&-labelled pig Z glycoproteins. )hese experiments" using Zs from either

follicular or o#ulated eggs" consistently re#ealed upta;e by a sperm protein of

Dr /$ ;,a that was



identied immunologically and biochemically as proacrosin" the zymogen form

of the serine protease acrosin (Gones and rown" >BHE Gones" >>E Irch and

atel" >>!. +ome binding was also obser#ed to a group of low Dr proteins in

seminal plasma" later identied as a family of seminal #esiclederi#ed

glycoproteins ;nown as sperm adhesins ()opfer-etersen et al." >>B!. i#en

the high degree of selecti#ity of the Z probe for proacrosin shown by thefailure of the probe to bind to the large number of other sperm proteins present

on the blot" the nature of the interaction was in#estigated further. )he current

state of ;nowledge may be summarized as followsJ . roacrosin-Z

glycoprotein binding is non-enzymic (Gones" >>E Gansen et al." >>/!. )he

inclusion of proteinase inhibitors (% mD 9,)A" / mD p-aminobenzamidine

(pA!" 88 g leupeptin! in incubation bu3ers has no e3ect on upta;e of the

probe (in fact" inhibitors consistently increase binding by 8/2!. &n any case"

proacrosin is inherently inacti#e. %. inding of K%/&LZs to puried proacrosin

shows a broad optimum between p0 /.8 and B.8 and is salt-dependent" with a

sharp maximum at /8 mD 6aCl (<illiams and Gones" >>$!E /82 inhibition

ta;es place at ?/8 mD 6aCl. )his suggests a strong ionic interaction stabilized

by co-ordinated hydrogen bonding between guanido moieties on sulphate

groups as described for bindin#itilline en#elope interactions (,e Angelis and

labe" >>8!. $. &nacti#e -acrosin and proacrosin are e@ually e3ecti#e in Z

binding assays (Gones" >>!. )he fact that proacrosin is con#erted to -acrosin

by internal clea#ages (aba et al." >B>! at both the 6-terminus (between

arginine %$ and #aline %?! and the C-terminus (arginine $%% and proline $%?!

suggests that neither the so-called 4light chain5 nor the proline-rich C-terminal

peptide are in#ol#ed in Z binding. )his has been conrmed by isolation of the

nati#e %$ residue light chain and articial synthesis of the peptide.

+ignicantly" polyarginine" polylysine and polyhistidine ha#e only wea; bindingcapacity as do %8-mer and $8-mer synthetic peptides of arginine (Gansen et al."

>>/!. )his is suggesti#e of a secondary structural re@uirement for Z binding

by the peptide (see below!. ?. )he mechanism of the interaction in#ol#es

recognition of polysulphate groups on Z glycoproteins by basic residues on the

surface of proacrosin in a manner similar to that for heparinantithrombin &&&

interactions (Gones" >>E Irch and atel" >>E Gin et al." >>H!. Contrary to

early claims" binding does not in#ol#e fucose and it is not a lectin-li;e

interaction. &n the pig" all three classes of Z glycoproteins are sulphated in

their carbohydrate moieties (6a;ano et al." >>8! and all three bind to

proacrosin. 0owe#er" carbohydrates per se are not re@uired for binding exceptinsofar as they pro#ide a repeating polymeric framewor; for presentation of

sulphate groups in the correct stereochemical alignment. :. 0owes" M. Gones '

Gournal of Meproductie &mmunology

)hus" some sulphated polymers (e.g. fucoidan" dextran sulphate /88 N"

galactan" poly#inylsulphate! are strong competitors of Z-proacrosin binding



whereas others (e.g. chondroitin sulphates" heparin! are #ery wea;

competitors. /. inding of sulphate groups re@uires the correct presentation of

positi#ely charged basic residues on the surface of the proacrosin. Chemical

modication of arginines" lysines and histidines with phenylglyoxal"

diethylpyrocarbonate or citraconic anhydride completely abolishes binding

(Gansen et al." >>/!. )hese reagents" howe#er" are not site-specic" but byexpressing deletion recombinants of boar proacrosin in bacteria it has been

possible to identify a minimum binding fragment represented by residues ?H to

%H% (Fig. %!. +ite-directed mutagenesis of selected amino acids within this

fragment has identied some of the critical residues in#ol#ed" such as histidine

?Harginine /8" / together with arginine %/8" lysine %/% and arginine %/$

(Gansen et al." >>B!. +ignicantly" substituting a cluster of lysines (H$"

H/"H1"HH! adPacent to histidine ?H has no e3ect on binding of Zs. Doreno and

arros (%888! found that a synthetic peptide representing residues /> to H1

inhibited Z glycoprotein binding to boar proacrosin" pointing to a role for

arginines 111Hhistidine 1>. &n rabbit acrosin" Michardson and O5Mand (>>1!

also deduced from mutagenesis studies that histidine ?H was important for Z

binding. )he abo#e residues" howe#er" are unli;ely to be the only ones in#ol#ed

in polysulphate binding as the stoichiometry of proacrosin-Z glycoprotein

binding is not ;nown. 1. )he disco#ery that suramin" a sulphonated anticancer

drug" is a highly e3ecti#e competitor of proacrosin-Z binding has pro#ided

insights into the stereochemical re@uirements of the reacting groups (Gones et

al." >>1!. A signicant property of suramin is that it does not penetrate cell

membranes and conse@uently does not bind to acrosome-intact spermatozoa.

+ince it is #ery e3ecti#e in bloc;ing fertilization in #itro" it seems li;ely that it

acts after the acrosome-reaction has ta;en place when the acrosomal contents

are exposed. &t has no apparent e3ect on sperm motility" nor does it induce theacrosome reaction. +uramin is now of considerable interest as a tool for

dissecting mammalian spermegg interactions. &ts principal merits areJ (i! it is a

small molecular weight compound (Dr ?%8! with a well-dened chemical

compositionE (ii! it has a symmetrical structure (Fig. $!" a property which seems

related to its ability to cross-lin; proteinsE (iii! sulphonate groups on the

terminal naphthalene ring structures are necessary for high a*nity bindingE

and (i#! a large number of analogues ha#e been synthesised (Gentsch et al."

>BH! enabling di3erent structural features to be in#estigated. As a result of

screening / of these analogues in the Z-proacrosin (boar! binding

assay" it was found that the parent compound presented the optimal

arrangement (Gones et al." >>1!. Meduction in the number of terminal

sulphonate groups or remo#al of any of the internal aromatic rings caused a

loss of competiti#e acti#ity. )he three-dimensional struc-ture of suramin is that

of a shallow ladle or cup (Adhi;eso#alu et al." >BB! suggesting that it is the

distance between the terminal sulphonated naphthalene rings that is the ;ey



feature for binding to" and cross-lin;ing" proteins. At the cellular le#el" suramin

is #ery e3ecti#e in inhibiting fertilization in #itro (Gones et al." >>1!. H.

&mmunodetection of proacrosin'acrosin on the inner acrosomal membrane and

on remnants of the acrosomal matrix following the acrosome reaction has been

demonstrated in the rabbit" hamster and guinea pig (arros et al." >>%!. &n the

rabbit especially" acrosin could be detected on %/2 of sperm reco#ered fromthe peri#itelline space (Qaldi#ia et al." >>>!" prompting speculation that these

may be the sperm that are able to bind to and fertilize fresh zona-intact eggs

(Nusan et al." >B?!. roacrosin is well ;nown to be an inherently 4stic;y5

molecule as it will

Fig. . roposed model of se@uence of e#ents during spermegg binding in

mammals. A capacitated sperm penetrates through the cumulus oophorus and

arri#es on the surface of the zona pellucida (step !. )he acrosome-intact

sperm binds to primary receptors (glycoprotein Z$J steps % and $! followed by

induction of the acrosome-reaction (step ?!. )his exposes the acrosomal matrix

and e#entually the inner acrosomal membrane. inding (step /! is maintained#ia a secondary Z receptor (glycoprotein Z%!. )he motile sperm begins to

penetrate through the Z and e#entually accesses the peri#itelline space where

it binds to a tertiary receptor (integrin'C,>! on the oolemma (reproduced from

Gones et al." >>1!. Fig. %. :inear distribution of basic and acidic amino acids in

the primary se@uence of boar proacrosin. (Meproduced from Gansen et al."

>>/!. Fig. ?. )hree-dimensional crystal structure of boar acrosin with

transposed model of suramin to illustrate relati#e molecular sizes. +ulphonate

groups on suramin are coloured orange (Acrosin structure deri#ed from )ranter

et al." %888!.

bind to glass surfaces" liposomes and cell membranes in general which induce

contact acti#ation (arrish et al." >HBE :o :eggio et al." >>?!. &t is not clear

what property is responsible for this apparent stic;iness as hydropathy plots do

not re#eal any pronounced hydrophobic peptide domains (aba et al." >B>E

Nlemm et al." >>8!. 6onetheless" there are strong indications that

proacrosin'acrosin has the potential to act as a 4bridge5 between cells. $.

+tructural features of sperm proacrosin i#en the importance of

stereocompatibility between sperm proacrosin' acrosin and polysulphate

groups on Z glycoproteins for binding" a crystallographic analysis of boar

acrosin was underta;en to determine the surface orientation of the important

residues mentioned abo#e. From %8 mg of highly puried nati#e protein"crystals were obtained that di3racted to %. A and from these data three-

dimensional structures were obtained by molecular replacement analysis

()ranter et al." %888!. <hen the molecule is rotated about the =-axis" it is

possible to demonstrate that the clusters of basic residues" pre#iously

identied by site-directed mutagenesis to be in#ol#ed in binding (see +ection %

abo#e!" appear on loops proPecting from the hydrophobic core of the protein

and are positioned to either side of the acti#e site (Fig. ?!. +ignicantly" the



intramolecular distance between the two most important clusters approximates

to the span between the naphthalene rings at the extremities of suramin (Fig.

$!" suggesting a mechanism for its competiti#e inhibition of acrosin acti#ity.

)hat is" it forms a bridge across the acti#e site pre#enting access of substrate.

Although suramin binds to a #ariety of di3erent proteins" it shows some

interesting discriminating features in relation to serine proteases. )hus"suramin does not inhibit trypsin acti#ity and has only a wea; e3ect on

chymotrypsin" but strongly inhibits neutrophil elastase (and acrosinE Cadene et

al." >>H!. &t is possible that li;e elastase" there is more than one suramin-

binding site on acrosin and further analysis will be re@uired to identify these. ?.

enetic e#idence for binding of Z% glycoprotein to sperm acrosin &n the pig

system described abo#e" it was found that all of the Z glycoproteins bound to

puried proacrosin'acrosin immobilized on dotblots. &n addition" F&)C-labelled

pig Zs bound more strongly to permeabilized boar sperm (Gones" >>! than to

intact cells suggesting that in its nati#e state within the acrosome" where it is

complexed to inhibitors and :. 0owes" M. Gones ' Gournal of Meproductie

&mmunology /$ (%88%! B>% B> #arious binding proteins"

proacrosin'acrosin could still interact with Zs. +ince there is good e#idence in

the mouse to suggest that the secondary receptor for sperm is Z% (leil et al."

>BB!" it may be predicted that Z% will bind to mouse acrosin in preference to

other sperm proteins. )o test this hypothesis" we ha#e compared binding of

mouse K%/&LZ% to wild-type ('! and acrosin ;noc;out (R'R! sperm using

light microscope autoradiography. &t was found that K%/&LZ% bound to

permeabilized but not to acrosome-intact sperm which is in agreement with the

obser#ations by leil et al. (>BB!. <hen permeabilized ('! and (R'R! sperm

were compared" there was an 1/2 decrease in the number of sil#er grains

associated with the head of acrosin-decient sperm. A similar gure wasobtained for upta;e of K$ 0Lsuramin. )his pro#ides the strongest e#idence so

far that proacrosin'acrosin is the maPor sperm ligand molecule for the

secondary Z receptor" Z%. )he small amount of binding of K%/&LZ% to

acrosin (R'R! sperm may be due to two other serine proteases that are maPor

components of the mouse acrosome (relati#e to other species" mouse sperm

are #ery decient in proacrosin!. )he fact that Z% binding to acrosin (R'R!

sperm is greatly reduced but not abolished concurs with the nding that

acrosin (R'R! sperm are capable of fertilizing eggs" albeit with reduced

e*ciency (aba et al." >>?E Adham et al." >>H!. 6onetheless" the ability of

K%/&LZ% and K$ 0Lsuramin probes to discriminate between acrosin ('! and(R'R! sperm can be attributed to their a*nity for proacrosin'acrosin. /.

Conclusions Currently" therefore" there are good reasons to belie#e that

proacrosin' acrosin has a role as a secondary binding molecule. &t is e3ecti#ely

targeted to its site of action following release from the acrosomal #esicle and

hence is present at the appropriate time and place to interact with the zona

surface. i#en the added physical constraints that surrounding cumulus cells

will ha#e on sperm mo#ements on the surface of the Z" the formation of ionic



bonds between proacrosin'acrosin and Z glycoproteins (in the mouse"

specically Z%! would be of su*cient tenacity to pre#ent the sperm from

detaching. )his would allow time for the sperm head to initiate zona

penetration. )he ability of the drug suramin to bind strongly to proacrosin'

acrosin and inhibit sperm-zona binding suggests that safe mimetics of the

compound ha#e potential as antifertility agents if released near the site offertilization" e.g. from long-acting &I,s. >8 :. 0owes" M. Gones ' Gournal of

Meproductie &mmunology /$ (%88%! B>% Ac;nowledgements <e than; the

+MC for nancial support. <e are also grateful to Gohn Coadwell (&! for

assistance with molecular modelling and numerous colleagues in the laboratory

and elsewhere who ha#e contributed to these studies o#er se#eral years.

%.AMC0&QO

Acrosin (9C $.?.%.8! is serine proteinase localized in the sperm acrosome and

considered to play an essential role in fertilization. &n contrast to mammalian"

there are only limited data concerning a#ian acrosin" mostly focused on the

characterization of mature enzyme. &n the present study" acrosin was isolated

from tur;ey spermatozoa using gel ltration in the presence of ? D urea at

acidic p0. 6-terminal 9dman se@uencing allowed the identication of the rst

%1 6-terminal amino acidsJ QQ)9A:0 +<<&Q+&S6MFA). )his se@uence

was used to construct primers and obtain a c,6A se@uence from the testis. )he

amino acid se@uence deduced from the c,6A shows that tur;ey acrosin is

initially synthesized as preproprotein with >-residue signal peptide. )his signal

se@uence is followed by a $%H-residue se@uence corresponding to the acrosin

zymogen. )ur;ey proacrosin does not contain a proline-rich segment at theCterminal portion. Dature tur;ey acrosin is a two-chain molecule consisting of

light and hea#y chains and was found to be glycoprotein. )he

proacrosin'acrosin system exists in tur;ey spermatozoa and this system can be

acti#ated similarly to that of mammals. )he high #alue of association constant

strongly suggests that acrosin acti#ity in tur;eys can be controlled by a seminal

plasma Nazal inhibitor under physiological conditions. 7 %88 9lse#ier &nc. All

rights reser#ed

. &ntroduction )he acrosome is a uni@ue #esicle surrounding the anterior

part of the spermatozoa head and contains a number of hydrolytic

enzymes" among which serine proteinase acrosin is the most studied

acrosomal enzyme (Chen et al." >>!. Acrosin is considered to play an

essential role in fertilization" recognition" binding and penetration of the

zona pellucida of the o#um (Nlemm et al." >>!. Acrosin is synthesized

and stored in the sperm acrosome as proacrosin" an inacti#e zymogen

form. ,uring the acrosome reaction" proacrosin is con#erted into mature

acrosin by a two part acti#ation processJ the liberation of proenzyme



segments from C-terminus and clea#age of a peptide bond producing the

light and hea#y chains (aba et al." >B>a!. )he role of acrosin in the

recognition and binding of oocytes en#elopes has ne#er been shown in

birds. &n contrast to mammalian" there are limited data concerning a#ian

acrosin. )he presence of trypsinli;e acti#ity in tur;ey and chic;en

spermatozoa extract has been demonstrated (Froman >>8E 0o andDeizelE >H1" Michardson et al." >BB!. )ur;ey acrosin was isolated and

partially characterized (Michardson et al." >BBE Michardson et al." >>%!.

&nhibition studies indicated that tur;ey acrosin amidase acti#ity was

inhibited by aprotinin" o#omucoid" soybean trypsin inhibitor"

benzamidine and zinc. A clinical method based on the determination of

sperm amidase acti#ity of sperm%. suspensions was optimized for tur;ey sperm (logows;i et al." %88!. At

present" there is no information concerning the proacrosin'acrosin

system of tur;ey spermatozoa" the structure of proacrosin protein and its

se@uence. ,i3erences in the e#olution of mammalian and bird protease

complement are mainly related to immunological" de#elopmental"

reproducti#e and neutral functions (Suesada et al." %88!. Meproduction

is one of the most dissimilar processes between birds and mammals.

Dultiple di3erences in protease gene in#ol#ed in fecundation and

embryo hatching are found. )estin serine proteinase and se#eral

members of the A,AD family of metalloproteases (A,ADs H and A,AD

$8! in#ol#ed in fertilization in mammals are lac;ing in the genome of

birds. On the other hand" acrosin is conser#ed in birds and mammals

(erlin et al." %88B!. )he genomes of human and chic;en contain a single

acrosin gene (ACM!" howe#er" the zebra nch genome contains se#en

non-clustered ACM-li;e genes. )herefore" studies concerning acrosincharacterization are necessary in order to dene species-specic

properties of #ertebrate acrosins. )ur;ey seminal plasma contains a low-

molecular weight trypsin inhibitor which was identied as a Nazal family

inhibitor" which can e3ecti#ely inhibit trypsin-li;e enzymes in #i#o

(+TowiUs;a et al." %88B!. Nazal inhibitors present in mammalian semen

are called acrosin inhibitors (:as;ows;i and Nato" >B8!. )hey are

present in a high concentration in seminal plasma and are also attached

to the spermatozoa surface (Gona;o#a et al." >>%!. )he main postulated

physiological function of Nazal inhibitors is the protection of reproducti#e

tissues" seminal plasma proteins or #iable spermatozoa from the

proteolytic action of acrosin$. liberated from acrosomes of dead and damaged spermatozoa (:as;ows;i

and Nato" >B8E :essley and rown" >HB!. &t is un;nownif Nazalinhibitors

from a#ian semen" similarly to its mammalian counterpart" control

acrosin acti#ity under physiological conditions. )he length of storage of

tur;ey semen is #ery restricted and usually after %? h of storage

signicant decreases in sperm #iability and fertilizing capacity are



obser#ed (Clar;e et al." >B%E ,onoghue and <ishart" %888!. A decline in

sperm @uality is accompanied by changes in phospholipids (due to their

lysis!" endogenous metabolism" or lipid peroxidation (,ouard et al."

%888E ,ouard et al." %88?!. &n our pre#ious wor;" we showed that a

decrease in tur;ey sperm @uality during shortterm storage is also related

to disturbances in sperm amidase acti#ity" presumably connected topremature acti#ation of acrosomal serine proteinases (NotTows;a et al."

%88H!. )herefore" it is possible that the decline in tur;ey semen @uality

and fertility is caused by uncontrolled proteolysis in spermatozoa.

Nnowledge concerning the characterization of the tur;ey

proacrosin'acrosin system and its control is re@uired for better

understanding of tur;ey semen physiology and the impro#ement of

li@uid semen storage. &n the present wor;" we demonstrate the presence

of the proacrosin' acrosin system in tur;ey spermatozoa. )he primary

structure of tur;ey proacrosin deduced from the nucleotide se@uence of

the cloned c,6A is also reported. hysicochemical characteristics of

tur;ey acrosin are described. 6aturally occurring seminal plasma

inhibitor can potentially control acrosin acti#ity under physiological

conditions. %. Daterials and methods %.. Animals and sampling +emen

was routinely collected by abdominal massage (urrows and Suinns"

>$H! from tur;eys (Deleagris gallopa#o! of I) ig-1 line (ritish

Inited )ur;eys :imited" Chester" 9ngland!" during the reproducti#e

season. )oms were maintained under standard husbandry conditions at

the )ur;ey )esting Farm in Frednowy" oland. ooled semen samples (8./

m:! were centrifuged at %"B88Vg for $ min to separate the

spermatozoa from seminal plasma. +permatozoa was subPected to

protein extraction using the method described by Michardson et al.(>BB!. First" sperm were washed three times with 8.BB2 6aCl and

centrifuged. 6ext" sperm pellets were than resuspended in 8./ m: of ? D

urea frozen in li@uid nitrogen" and thawed two times. +perm suspensions

were then placed in an ice bath" sonicated for $8 s with QC-$

(+onics" I+A!" set at $/2 relati#e output" and centrifuged for $ min at

%"B88Vg. )he sperm extract was prepared immediately before

purication on +uperdex %88 W'N 0i:oad 1'18 column (Amersham

ioscience" Ippsala" +weden!. Fresh testes designed for c,6A cloning

were obtained from tur;eys of the I) ig-1 line and immediately frozen

in li@uid nitrogen and stored at RB8 XC. Appro#al from the Animal9xperiments Committee in Olsztyn" oland was obtained before starting

any experiments. %.%. Acti#ation of serine proteinase of tur;ey

spermatozoa +perm extract was prepared in ? D urea as described

pre#iously and acti#ated at $H XC in 8.$ D )ris0Cl" p0 B.8 for ? h. At ten

minute inter#als samples were ta;en and subPected to an amidase

acti#ity measurement and to gelatinolytic +,+-A9 electrophoresis

under non-reduction condition. enzamidine was added to a



concentration of %8 mD to samples for +,+-A9-gelatinase in order to

stop serine proteinase acti#ation. %.$. urication of acrosin from tur;ey

spermatozoa )he freshly prepared sperm extract ( m:! was ltered

through a 8.?/ Ym pore size syringe lter and further fractionated by gel

ltration

?. (F! chromatography on +uperdex %88 W'N 0i:oad 1'18 column.roteins were eluted with ? D urea" p0 $.8 at m:'min using an F:C

system (Amersham ioscience!" and detected at %B8 nm. )he obtained

fractions ( m:! were screened for acrosin amidase acti#ity and

electrophoretic detection of gelatinase acti#ity. Acrosin-containing

fractions were subPected to measurement of the e@uilibrium association

constant for acrosininhibitor complexes and autoacti#ation. Additionally"

acrosin fractions obtained after gel ltration were puried using re#erse

phase chromatography (MC! in the 0:C system. )he lyophilized

fractions of acrosin were dissol#ed in 8.2 triuoroacetic acid ()FA"

Flu;a" uchs" +weden!" ltered through a 8.%% Ym pore size syringe lter

and further puried on ioasicB column ()hermo 0ypersil-Neystone"

Muncorn" Inited Ningdom! e@uilibrated with 8.2 )FA. ound proteins

were eluted with a linear gradient (82H/2! of B82 acetonitrile (Derc;"

,armstadt" ermany! and 8.8H2 )FA (ow rate m:'min!. )he acrosin

was eluted at a range of ?B2/2 acetonitrile. )he eluted pea;s were

lyophilized. Fractions obtained after MC were used for the determination

of 6-terminal se@uences of acrosin and mass spectrometry. %.?.

Autoacti#ation of gel ltration fractions )he F fractions were screened

for proacrosin by acti#ation to acti#e proteinases. Autoacti#ation was

performed by incubation at $H XC for $ and %? h at p0 B.8. After

acti#ation samples were ta;en and subPected to amidase acti#itymeasurement and to gelatinolytic +,+A9 electrophoresis under non-

reduction condition. &ncrease of amidase acti#ity was expected as

indicator of proacrosin acti#ation. %./. Analytical methods )he protein

concentration was measured by :owry et al. (>/! and radford (>H1!

using reagents from +igma-Aldrich Chemicals Co. (+t :ouis" DO" I+A!

and bo#ine serum albumin as the standard. +perm amidase acti#ity was

measured according to the method described by eiger and Fritz (>B$!

with modications described by Cieresz;o et al. (>>1a" >>1b!" using 6-

[-benzoyl-,:-arginine-p-nitroanilide (A6A! as a substrate. One unit (I!

of amidase acti#ity is dened as the amount of enzyme that hydrolyzes

\mol :R A6A'min at %/ XC. 9lectrophoretic detection of gelatinase

acti#ity was performed according to themethod of +iegel and ola;os;i

(>B/! as pre#iously described (NotTows;a et al." %88/" %88H!. Ali@uots

(8.% \g protein! of sperm extract were loaded into each well (Fig. !.

After electrophoresis" the gels were washed at room temperature with

%./2 )riton W-88 for ?8 min" incubated in de#elopment solution (/8 mD

)ris0Cl bu3er p0 H./" containing %88 mD 6aCl" 8.8%2 )riton W-88! for



h at $H XC. rotein bands after F were identied by +,+-A9 under

non-reduction conditions on 82 slab gel (:aemmli" >H8!. Ali@uots (/8

\g protein! of sperm extract and / \g protein of F fraction were

loaded into each well (Fig. %!. Antitrypsin acti#ity was e#aluated by the

inhibition of bo#ine trypsin amidase acti#ity according to the method of

eiger and Fritz (>B$! with modications pre#iously described(Cieresz;o et al." >>1a" >>1bE Cieresz;o et al." >>B!. Antitrypsin

acti#ity after A9 (82 polyacrylamide gel! was identied using bo#ine

trypsin (+igma! according to Iriel and erges (>1B! as described

pre#iously (NotTows;a et al." %88/!. Ali@uots (%8 \g protein! of seminal

plasma and H \g protein of F fraction were loaded into each well (Fig.

$!. Following electrophoresis" the gels were incubated at $H XC for /

min with bo#ine trypsin in 8. D phosphate bu3er (p0 H.?! and then

transferred into a solution containing a chromogenic substrate (acetyl,:-

phenylalanine-]-naphthyl ester! for trypsin. +tained gels were stored in

%2 acetic acid. )he zones possessing inhibitory acti#ity against the

chosen enzymes appear as unstained bands on a coloured bac;ground./. %.1. ,etermination of 6-terminal se@uence of acrosin )he concentrated

fractions of acrosin after MC were dissol#ed in 0:C grade water

(+igma!. +amples were boiled and subPected to tristricine +,+-A9

(+chagger and #on Gagow" >BH! and electroblotted onto a Q,F

membrane using 8 mD $-(cyclohexylamino!--propanesulfonoic acid-

6aO0 (p0 .8! containing 82 methanol. )he membrane was stained

in 8.2 Coomassie rilliant lue M-%/8 in ?82 methanol and 2 acetic

acid and destained in /82 methanol. 6-terminal protein se@uence

analysis was performed at ioCentrum :td. (Cracow" oland!. )he

se@uentially detached phenylthiohydantoin deri#ati#es of amino acidswere identied using the rocise ?> (Applied iosystems" Foster City"

CA" I+A! automatic se@uence analysis system" according to standard

protocol of the manufacturer. +e@uence comparisons were performed

using the database +<&++rot (httpJ''www.ncbi.nlm.nih.go#'blast!. %.H.

Cloning and se@uencing of proacrosin )he fresh tur;ey testes were cut

into small pieces and immediately frozen in li@uid nitrogen. )he tissue

was grinded in li@uid nitrogen to a ne powder with a mortar and pestle

followed by 6ucleo+pin lter homogenization (Dacherey-6agel"

ermany!. )otal M6A from approximately ?8 mg of disrupted tissue was

puried using 6ucleo+pin M6A && Nit (Dacherey-6agel!. )he @uantity and

integrity of the total M6A were chec;ed by formaldehyde agarose gel

electrophoresis. )o identify the proacrosin c,6A" a set of #e degenerate

and nondegenerate primers were designed based on the partially ;nown

polypeptide se@uence of the protein and acrosin-related proteins from

the 9D: database. )he c,6A was synthesized using +uperscript &&&1. re#erse transcriptase (&n#itrogen" I+A! with poly-d)(%8! primer using /

Yg of total M6A as a template. From six reactions which occurred in the



experiment" the pair of DO-F primer and DO%-M primer ()able ! ga#e a

dominant CM product of /H1 bp. )he product was puried from the

agarose gel" cloned into the pCM?-)OO #ector" and se@uenced. All CM

reactions were performed with Fast+tart 0igh Fidelity )a@ olymerase

(Moche" ermany!. )he /′ and $′ MAC9 was performed according to

Frohmann (>>$!. ased on the partial se@uence of proacrosin" a set of/′ MAC9 re#erse primers and $′ MAC9 forward primers were designed

()able !. )he synthesis of c,6A from / \g of the total M6A was

performed using the Acro/ primer in /′ MAC9 and adaptor-oligo-d)(H!

primers in $′ MAC9. After the /′ MAC9 M) reaction" primers" nucleotides and

salts were remo#ed by ultraltration on Dicrocon-88 columns (Dillipore"

I+A!" with sterile miliS-grade water for dilution. )he puried c,6A was

tailed using terminal transferase (romega" I+A! in the presence of 8.%/

mD dA) at $H XC for 8 min. )he second strand of c,6A was

synthesized using adaptor-d)(H! primer. After the addition of adaptor

and Acro/% primers" the rst /′ MAC9 reaction was performed" followed by

^semi-nested_ CM using the Acro/$ primer and adaptor primer. )he

discrete product was puried and cloned into the pCM?-)OO se@uencing

#ector. )he $′ MAC9 product was amplied after the addition of Acro$

primer and adaptor primer to $ ′ M) c,6A. )hen the Acro$% primer and

adaptor primer were used in ^semi-nested_ CM. )he specic product

was puried and cloned into the pCM?-)OO #ector. All ,6As were

analyzed by se@uencing (+e@uencing :aboratory" &nstitute of

iochemistry and iophysics" olish Academy of +ciences" <arsaw"

oland!. )he translation of a nucleotide se@uence to a protein se@uence

was performed using the )ranslate rogram (httpJ''us.expasy.org'tools'

H. dna.html!. )he putati#e signal peptide was determined using the +ignal$.8 program (httpJ''www.cbs.dtu.d;'ser#ices'+ignal'! according to

6ielsen et al. (>>H!. Dolecular weight and isoelectric point of the

translated protein were calculated using the rotaram )oll program

(httpJ''us.expasy.org'tools'protparam.html!. osttranslational

modication prediction of 6-" O-glycosylation sites was determined using

the 6et6lyc .8 +er#er according to upta et al. (in preparation!

(httpJ''www.cbs.dtu.d;'ser#ices'6et6lyc'! and 6etOlyc $. +er#er

according to Gulenius et al. (%88/! (httpJ''www.cbs.

dtu.d;'ser#ices'6etOlyc'!. otential clea#age sites clea#ed by trypsin

were predicted using eptide Cutter according to asteiger et al. (%888!(httpJ''www.expasy.ch'tools'peptidecutter'!.B. %.B. ,etermination of molecular mass Dolecular mass was determined

using mass spectrometry carried out on a ru;er 9s@uire $888 lus

9lectrospray &onization (9+&! ion trap mass spectrometer (ur;er-

,altonics" remen" ermany! at the Megional :aboratory of

hysicochemical Analyses and +tructural Mesearch (Cracow" oland!. %.>.

,etermination of isoelectric point )he isoelectric point was determined

http://us.expasy.org/tools/

http://www.expasy.ch/tools/peptidecutter/

http://www.expasy.ch/tools/peptidecutter/

http://us.expasy.org/tools/



by the isoelectric focusing (&9F!. )he isoelectric focusing of acrosin was

carried out under non denaturing conditions in ready &9F gels with a p0

range of /B with cathode and anode &9F bu3ers (io-Mad" 0oefer" +an

Francisco" CA" I+A! for %./ h in total" starting with constant #oltage of

88 Q for h"

>. then increasing to %/8 Q for the next h and to /88 Q for $8 min. For p&calibration the &9F mar;er ;it (io-Mad! was used. &soelectric point of

acrosin was estimated with the use of the Noda; , program (9astman

Noda; Company" 6ew 0a#en" C)" I+A!. %.8. ,etection of glycan moiety

lycoprotein staining was performed after +,+-A9. )he gel was

stained with lycorole &&& staining solution according to the

manufacturer`s protocol (+igma!. %.. Deasurement of the e@uilibrium

association constant (Na! for the acrosinNazal inhibitor complex Acrosin-

containing fraction after F was concentrated using I)ube Concentrators

(Dillipore! to /888 I'm: of amidase acti#ity. )he acrosin concentration

was calculated by acti#e site titration with pnitrophenyl-p′ -

guanidinebenzoate (6! using the same procedure as for trypsin

titration (Chase and +haw" >1H!. &t appeared that ? D urea present in

the titration sample interacting with 6 causes a decrease in

calculated enzyme concentration. Acti#e site titration of bo#ine trypsin in

the presence of urea showed a loss of trypsin concentration of about

/82 (data not shown!. )herefore we increased the obtained acrosin

concentration in the presence of urea by /82. )he concentrations of

Nazal inhibitor from seminal plasma were determined by titration with

titrated bo#ine trypsin. Nazal inhibitors used for the measurement of Na

were isolated from tur;ey seminal plasma according to +TowiUs;a et al.

(%88B!. )he e@uilibrium association constant (Na! #alues weredetermined in 8. D )ris0Cl" 8.88/2 )riton W-88" p0 B.$ at %/ XC using

the method of reen and <or; (>/$! as described by 9mpie and

:as;ows;i (>B%!. )he enzyme concentrations K98L used complied with

the e@uation %bK98L VNab/8" and the concentrations of inhibitors K&8L

ranged from 8 to %V K98L. &ncreased amounts of inhibitors were added to

a constant concentration of trypsin and after / min of incubation the

residual enzyme acti#ity was measured using a turno#er of the substrate

6-[-benzoyl-,:-arginine-p-nitroanilide (+igma!" whose nal

concentration in the reaction medium did not exceed 8.% Nm. Ninetic

measurements were made at ?8/ nm for %8 s. %.%. Meplicates and

statistical analysis )he #alues Na were calculated by three-parameter

algorithm K9L f(98" Na" F! using the non-linear regression analysis

program raFit $.8 (9rithacus +oftware :td" Diddlesex" 9ngland!"

according to the e@uationJ 9 where K98L and K&8L are the total

enzyme and inhibitor concentrations" respecti#elyE K9L is the residual

enzyme concentration" andF is the enzyme inhibitor e@uimolarity factor.

)his experiment had been replicated independently three times.



Acti#ation experiment had been replicated two times. All biochemical

measurements were made in duplicate. $. Mesults $.. 93ect of

incubation time on amidase acti#ity and geltinases of tur;ey sperm

extract &ncubation at p0 B.8 induced increased amidase acti#ity of

tur;ey sperm extract (Fig. A!. )he highest amidase acti#ity was found at

8 min of incubation and the acti#ity at the time point wasapproximately % times higher than at time 8. )he changes of

electrophoretic proles of gelatinolytic acti#ity at the same time are

shown inFig . )hree forms8.forms of gelatinase were present in the sperm extract at time 8. After 8

min of incubation one additional band of molecular mass %B ;,a

appeared and the gelatinase with a molecular mass of $8 ;,a seemed to

increase its intensity. <e assumed that the band of molecular mass of $8

;,a represented an acti#e form of acrosin and was subPected to further

purication (see below!. )he gelatinase of a molecular mass of $$ ;,a

gradually disappeared during the incubation time (Fig. !. )he intensity

of the ? ;,a gelatinase seemed to decrease with time but did not

disappear completely during the incubation time. All gelatinases were

inhibited by benzamidine (data not shown!. $.%. &solation of acrosin from

tur;ey spermatozoa and identication of Nazal inhibitor in sperm extract

)he one-step isolation procedure led to a /.B-fold increase of puried

acrosin with a yield of %?2. el ltration in acidic conditions was

necessary to limit the acti#ation of proacrosin and continuous generation

of intermediate forms of acrosin during the chromatography process.

)his procedure allowed us to separate acrosin from most sperm proteins

and inhibitors (Fig. %A" !. After gel ltration we obtained two fractions of

amidase acti#ity (Fig. %A!. )he rst fraction (F&! was characterized bylower amidase acti#ity" and the second fraction (F&&! was characterized

by three-fold higher amidase acti#ity than that of F& and contained a

dominant protein band (lanes HHB$ in Fig. %! which was later

conrmed to be acrosin. )herefore" F&& was used for further

physicochemical and ;inetic analysis. After F&& the pea; of antitrypsin

acti#ity was eluted (see below" Figs. %A and $!. )he fraction of antitrypsin

acti#ity contained two inhibitors of moderate and fast migration rate that

could be #isualized by re#erse zymography. )he migration rate of

characterized inhibitors was the same as Nazal inhibitors from tur;ey

seminal plasma (Fig. $!. $.$. Acti#ation of fractions obtained after gel

ltration Fractions & and && obtained after gel ltration were subPected to

acti#ation. F& contained two #ery intensi#e proteolytic bands with

molecular masses of $$ and ? ;,a. F&& contained additional bands with

a molecular mass of $8 ;,a. After acti#ation in F&" two bands of

proteolytic acti#ity were obser#edJ bands with molecular masses of $8

and ? ;,a. Only the band with a molecular mass of $8 ;,a was #isible

in F&& after acti#ation (Fig. ?!. 6o increase in amidase acti#ity was found



in F& and F&& after acti#ation (data not shown!. $.?. Amino acid

se@uencing 6-terminal 9dman se@uencing of the main protein of F&&

allowed us to identify the rst %1 6-terminal amino acidsJ

QQ)9A:0+<<&Q+&S6MFA). )his se@uence shows a high similarity

to a#ian and mammalian acrosin (see ,iscussion!. $./. Complementary

,6A cloning )he identied %1 amino acid se@uence of acrosin was usedto construct primers and obtain a c,6A se@uence from the tur;ey testis

(Fig. /!. )he nucleotide and amino acid se@uences were deposited in the

9D: 6ucleotide +e@uence ,atabase under accession number

AD1H>H? and CAG?/8%H" respecti#ely. )he full-length c,6A of the

acrosin was %?8 bp long" including a / ′ -terminal untranslated region

(I)M! of %8 bp" a $′ -terminal I)M of H> bp with a canonical

polyadenylation signal se@uence AA)AAA and poly(A! tail (Fig. /!. A

putati#e A) codon and a stop codon )AA were located at nucleotides %

and 8/>" respecti#ely. )he open reading frame is predicted to encode a

protein of $?1 amino acids with a calculated molecular mass of $H">/8.

,a and p& H.18. 0ydrolysis of a putati#e signal peptide (most li;ely

clea#age site between positions > and %8J 0-F+! should produce a

mature protein containing $%H amino acids" consisting of light and hea#y

chains (residues.% and %%$%H" respecti#ely! with a calculated molecular mass of

$1"8/H ;,a and p& H.$$. One potential 6-glycosylation site is present at

Asn %B and three potential O-glycosylation sites are present at )hr B?"

)hr $88 and )hr $%. $.1. 0omology analysis of tur;ey acrosin )he

se@uence of tur;ey acrosin was #ery similar to a#ian proacrosin c,6AJ

guinea fowl (6umida meleagris!" common @uail (Coturnix coturnix!"

mallard duc; (Anas platyrhynchos!" B?2" 1?2 and 1%2 identity"respecti#ely (Fig. 1!. A ??2?H2 identity was found to the acrosins of

the elephant shrew (Dacroscelides proboscideus!" llama (:ama glama!"

elephant (:oxodonta africana" 9lephantulus rufescens! and antelope

acrosin ()ragelaphus angarii!. A ?/2 identity was found in human

acrosin (0omo sapiens!. )hese identities include the acti#e site region"

substrate binding site and the location of cysteine. $.H. hysicochemical

characteristics )he molecular mass of acrosin estimated by mass

spectrometry was $8"BH? ;,a. )he isoelectric point of acrosin estimated

with isoelectric focusing was determined to be 1.?. Acrosin was stained

positi#ely for the presence of carbohydrates with a uorescent

glycoprotein detection ;it (data not shown!. $.B. )he e@uilibrium

association constant (Na! for acrosinNazal inhibitor complex )he

inhibition of tur;ey sperm acrosin by Nazal seminal plasma inhibitor is

shown in Fig. H. )he association constant (Na! was determined to be

H.1V8H DR8.1/. ?. ,iscussion &n the present study" acrosin from

tur;ey spermatozoa was puried and characterized. <e identied the

rst %1 6-terminal amino acids of a hea#y chain of acrosin and then used



this information to construct primers and obtain c,6A of a#ian

proacrosin for the rst%.time. )he tur;ey sperm proacrosinacrosin system was identied and

physicochemical characteristics of acrosin were obtained. )he high #alue

of e@uilibrium association constant for the acrosin'seminal plasma Nazal

inhibitor complex was determined which indicates that tur;ey acrosincan be e3ecti#ely inhibited by a naturally occurring seminal plasma

inhibitor as described by +TowiUs;a et al. (%88B!. )he extraction of

acrosin from tur;ey spermatozoa appeared to be more di*cult compared

with standard procedures for mammalian sperm. Irea and sonication

were necessary to extract acrosin from tur;ey spermatozoa (Michardson

et al." >BB!. A di*cult extraction of acrosin from tur;ey spermatozoa

li;ely reects tur;ey sperm physiology and indicates that acrosin is

strongly attached to the spermatozoa which was already indicated by

Michardson et al. (>BB!. 9xtraction of tur;ey spermatozoa with ? D urea

allowed us to obtain high le#els of amidase acti#ity (/888 I'l!.

Michardson et al. (>BB! suggested that this method of extraction

resulted in acrosin in its acti#e form but not as a zymogen.

Conse@uently" these authors concluded that acrosin is present in tur;ey

sperm in the acti#e form. 0owe#er" contrary to their wor; we were able

to acti#ate the proteolytic system of tur;ey sperm extracts and

demonstrate the presence of bands corresponding to proacrosin (? ;,a!

and the intermediate forms of acrosin ($$ ;,a!" ]-acrosin ($8 ;,a! and

brea;down products of ]-acrosin (%B ;,a!. &n conclusion" our results

strongly suggest that the proacrosinacrosin system exists in tur;ey

spermatozoa and this system resembles mammalian proacrosin

acti#ation (aba et al." >B>aE rown and 0artree" >HBE 9berspcher etal." >>E Deizel and Du;erPi" >H/E Deizel and Du;erPi" >H1E ola;os;i

and arrish" >HH!. )his conclusion is supported by c,6A se@uence

analysis (see below!. Our study demonstrated that in addition to acrosin

serine proteinase another enzyme is present. Our study also

demonstrated that other than acrosin serine proteinase is present in

tur;ey sperm extract. )his proteinase was represented by a band with a

molecular mass of ? ;,a (similar to proacrosin!. <e were not able to

acti#ate that proteinase with the procedure which was e3ecti#e for

acrosin of fraction &&. )his suggests that this protease represents an

enzyme di3erent than acrosin serine proteinase of tur;ey spermatozoa.

A number of acrosomal serine proteinases other than acrosin are present

in #arious mammalian sperm (0onda et al." %88%!. +ome of them ha#e

been isolated and$.characterized" such asJ bo#ine sperm protease" testicular serine

proteases" C)A-extracted sperm protease" and sperminogen (A;ama et

al." >>?E Cesari et al." %88/E Nohno et al." >>BE =u and =i" %88!. )he

biological role of these enzymes has not yet been identied. Future



studies should focus on the identication of the abo#ementioned serine

proteinase from tur;ey sperm extract and the establishment of its

relationship to the acrosin.?.&n our study the gel ltration fractions after acti#ation did not show an

increase in amidase acti#ity although con#ersion to mature form of

acrosin was obser#ed (Fig. ?!. )hese results suggest that either tur;eyproacrosin was inacti#ated at acidic p0 or most proacrosin acti#ation

occurred during the extraction and preparation of the extract to gel

ltration. For this reason isolation methods applied for mammalian

acrosin could not be used for tur;ey proacrosin. 0owe#er" gel ltration at

acidic p0 seemed to be an e3ecti#e method for the isolation of ]-acrosin

from tur;ey sperm extract. Acidic p0 also limited the generation of

multiple forms of an enzyme. )he one-step isolation procedure was

e*cient and produced the acrosin preparation as a one protein band

during +,+-A9 electrophoresis. )he se@uence of tur;ey acrosin was

homologous to a#ian c,6A se@uences attributed to proacrosin protein

(accession no. AS$>>>BE AS$>>>>E AS?888!. &nformation concerning

the characteristic of a#ian proacrosin protein is not a#ailable which limits

comparati#e analysis of our results. 0owe#er" the tur;ey acrosin

re#ealed a high identity to mammalian acrosin. )hese identities include

highly conser#ed se@uences surrounding the acti#e site region" substrate

binding site and the location of cysteines. )his allows the analysis of the

tur;ey acrosin se@uence on the basis of the structure of mammalian

acrosins as described by aba et al. (>B>a!. &t is li;ely that tur;ey

acrosin" similar to mammalian acrosin" is composed of light and hea#y

chains. 6-terminal 9dman se@uencing of tur;ey acrosin showed a high

similarity to the hea#y chain of mammalian acrosin (Fig. 1! and pro#ideddirect e#idence for the presence of the hea#y chain in tur;ey acrosin.

)he analysis of the tur;ey acrosin c,6A se@uence indicates that ahead

of the obtained 6terminal se@uence of the hea#y chain there are

additional % amino acids with a stri;ingly similar pattern of se@uence

homology to mammalian acrosin around the cysteine residue CysB-ly-

:eu-Arg and Arg %(Fig 1!. )his suggests that tur;ey acrosin similar

to the mammalian acrosin is a two-chain molecule consisting of the

light and hea#y chains (residues % and %%$%H" respecti#ely!. &t can

be assumed that the light chain is synthesized as an 6-terminal

extension of the mature acrosin and is separated from the hea#y chain

during the acti#ation of the zymogen. &t is li;ely that the light chain

remains co#alently attached to the hea#y chain by two disulde bonds.

)he location of cysteines forming disulde bonds between light and

hea#y chains is homologous to mammalian acrosin and suggests that

two cysteines in the light chain at residues / and B form the interchain

disulde bonds with two cysteines in the hea#y chain probably

residues %H and $/. &n the hea#y chain of acrosin se#eral



characteristic residues can be identied. )he 6-terminal se@uenceJ

QQ" is indicati#e of an acti#ated serine proteinase (aba et al."

>B>a!. )he proteolytic acti#e site segments 0is" Asp and +er are located

in this chain at positions 11" / and %/" respecti#ely. )he Asp residue"

which acts as a recognition site for the hydrolysis of peptide bonds

containing Arg or :ys (aba et al." >B>a!" is present at residue %8>. Fourinterchain disulde bonds are probably formed within the hea#y chain

between appropriate cysteine residues (between residues / and 1H"

?> and %%" B? and %88" and % and %?!. Cys $8 is probably

remo#ed during the maturation of the acrosin. +umming up" highly

conser#ed se@uences surrounding the acti#e site region" substrate

binding site and the location of cysteines are present in the hea#y chain

of tur;ey acrosin. )he maturation mechanism of acrosin zymogen"

particular boar proacrosin" has been well described for mammals (aba

et al." >B>a!. oar proacrosin is con#erted to mature acrosin by the

clea#age of the Arg%$Qal%? bond after the remo#al of the C-terminal

?-residue segment" resulting in the formation of the light and hea#y

chains. )his two-chain molecule is then con#erted to the mature enzyme

by remo#al of the C-terminal B- and ?$-residue segments. )he clea#age

sites in the C-terminal segment are localized at residue :ys$1/ Arg$11

and Arg$B$+er$B?. )hese clea#ages could be catalyzed by/.acrosin and'or trypsin. Only clea#age at residue Arg$%%ro$%$" the last

stage of acti#ation" can be carried out by acrosin" since trypsin is

incapable of splitting this peptide bond. A high degree of se@uence

similarities between tur;ey and mammalian proacrosin permits us to

predict both 6- and C-terminal clea#age sites of tur;ey proacrosin during

the maturation process. )he 6-terminal1.clea#age is located between Arg% and Qal%% as we described earlier.

)his clea#age would generate a %-residue light chain and much longer

hea#y chain. Also" there are#e potential clea#age sites in the C-terminal

portion of tur;ey proacrosin clea#ed by trypsin (:ys$%1Ala $%H" Arg

$8> lu$8" :ys$8H:eu$8B" Arg %H$)hr%H? and Arg%1/Ala%11!. )he

mentioned clea#age sites are di3erent from those described for

mammalian proacrosin. &t is unclear at present which of these bonds are

clea#age during acti#ation. 0owe#er" we also identied the clea#age

bond Arg%?1ro%?H" whose clea#age was the last acti#ation stage of

boar proacrosin. )herefore" the maturation of tur;ey acrosin as well as

that of mammalian would be accomplished by the clea#age of the Arg

ro bond. Further studies are necessary to pro#ide detailed information

concerning the tur;ey proacrosin acti#ation pathway. &nformation

presented abo#e indicated a stri;ing similarity between the general

structure of tur;ey and mammalian proacrosinacrosin systems. )here

are pronounced di3erences in reproduction biology between a#ian and

mammals and it is strange that these di3erences are not reected in the



structure and acti#ation of the proacrosin system. 0owe#er" the

similarity between tur;ey and mammal acrosins agrees with the

suggestion by erlin et al. (%88B! that the molecular e#olution of

gamete-recognition genes among birds and mammals is not dramatically

di3erent. As established abo#e" mature tur;ey acrosin should contain

%?1 amino acids in two chains" the %-residue light chain and %%/-residue hea#y chain. )he isoelectric point and molecular mass of tur;ey

acrosin calculated from the se@uence were 1.? and %1.>?>/ ;,a"

respecti#ely. )he isoelectric point estimated through isoelectric focusing

(1.?! was in agreement with the calculated p& #alue. )he molecular mass

of acrosin measured by mass spectrometry ($8"BH? ;,a! was $.>%? ;,a

higher than the molecular mass calculated from the se@uence. )his

discrepancy li;ely results from the glycoprotein structure of acrosin

which was found in this study and has been also demonstrated by

Michardson et al. (>BB! for tur;ey acrosin. )he analysis of c,6A

se@uence indicated that tur;ey acrosin contains one potential 6- and one

potential O-glycosylation sites. Further studies are necessary to

characterize the glycoprotein nature of tur;ey acrosin regarding the

structure of the carbohydrate chain and its connection to the protein

chain of the acrosin molecule.H.A characteristic feature of mammalian proacrosin is the presence of the

proline-rich segment localized at the C-terminal portion (aba et al."

>B>aE aba et al." >B>b!. )he functions of proline repeats ha#e not

been well dened. aba et al. (>B>a! suggested that the presence of

the proline-rich segment at the C-terminal portion of proacrosin may be

related to a high degree of hydration necessary for a high concentration

of proacrosin (.1 mD! in the boar acrosome. )he proline-rich segment issplit o3 during the con#ersion of proacrosin to mature form of acrosin. &t

is interesting that tur;ey proacrosin does not contain proline-rich

segment. roacrosin c,6A se@uences of other a#ian species also do not

contain proline repeats at the C-terminal portion (accession no.

AS$>>>BE AS$>>>>E AS?888!. As Materman and +pringer (%88B!

suggested" exon / codes for proline-rich region may be subPected to a

di3erent selection regime than the rest of the protein. &t is possible that

this region is e#ol#ing rapidly while the acti#e site region and the

location of cysteines are conser#ed. A lac; of the proline-rich segment

localized at the C-terminal portion of a#ian acrosin seems to be distinct

for a#ian acrosin. )he signicance of this phenomenon is uncertain at

present. )ur;ey seminal plasma contains a low molecular weight trypsin

inhibitor which was identied as a Nazal family inhibitor (+TowiUs;a et al."

%88B!. )he mechanism of the Nazal inhibitor'enzyme interaction is well

described and is termed a 4standard5 mechanism (:as;ows;i and Nato"

>B8!. )he inhibitor'proteinase complex could dissociate to the enzyme

and inhibitor in one of two formsJ #irgin and modied (a split peptide



bond!. As a result of split peptide bond" the modied form of inhibitor

di3ers from the #irgin form in higher molecular mass (by B ,a!" more

negati#e charge at high p0 and slower reaction with proteinase

(:as;ows;i and Nato" >B8E +TowiUs;a et al." %88B!. )he presence of

inhibitor in tur;ey seminal plasma in two forms" #irgin and modied (a

split peptide bond!" is a sign of its role in regulating the proteolyticprocesses in #i#o. &n this study we demonstrated for the rst time the

presence of a Nazal inhibitor in the tur;ey sperm extract. &t is un;nown if

this inhibitor can be attached to the spermatozoa surface or located in

the sperm. el ltration at acidic p0 allowed us to dissociate the Nazal

inhibitor'proteinase complex and identify two forms of Nazal inhibitor in

the sperm extract. )he presence of two forms of Nazal inhibitor in the

sperm extract is a sign of its acti#e role in the control of proteolytic

processes in spermatozoa. According to :as;ows;i and Nato (>B8!"

extremely high #alues of Na ranging between 8H and 8$ DR

indicate that the proteinase inhibitor can e3ecti#ely inhibit target

proteinase. )he association constant (Na! determined for the Nazal

inhibitor'acrosin interaction was H.1V8H DR . )his #alue was within

the high of Na #alues suggesting that a Nazal inhibitor can e3ecti#ely

inhibit acrosin in #i#o. )his result strongly suggests that the physiological

function of the Nazal family inhibitor of tur;ey semen is the inacti#ation

of acrosin released from dead or damaged and'or control of acti#ation of

the proacrosinacrosin system. &n conclusion" we ha#e demonstrated

that the proacrosin'acrosin system exists in tur;ey spermatozoa and this

system resembles mammalian proacrosin acti#ation. A characteristic

feature of tur;ey proacrosin is a lac; of a proline-rich segment at the C-

terminal portion. Dature tur;ey acrosin is a two-chain moleculeconsisting of the light and hea#y chains and was found to be

glycoprotein. )he association constant determined for Nazal

inhibitor'acrosin interaction was H.1 V 8H DR which suggests that

acrosin acti#ity can be controlled by a seminal plasma Nazal inhibitor

under physiological conditions.

abstract fertilization is one of the most specific and carefully regulated cell

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