Evidence for boar sperm proacrosin as a carbohydrate binding protein

Cell Biology

Evidence

international

Reports,

for boar sperm proacrosin

Vol. 11, No. 11, November

as a carbohydrate

R. Jones, Department of Molecular Embryology, Institute Genetics Research, Babraham, Cambridge CB2 4AT, U.K.

binding

1987

a33

protein

of Animal

Physiology

and

Recent investigations on fertilization in manuals have shown that carbohydrate moieties on cell surface glycoconjugates play an important role in spermegg recognition (1). Inhibition of fertilization with lectins and haptens have long implicated the presence of a ligand molecule on spermatozoa that binds to carbohydrates on the zona pellucida (2). Several putative ligands have been proposed e.g galactosyltransferase on mouse spermatozoa (3) and a fucosebinding protein on boar spermatozoa (4). Recently, we described a major zona-binding protein on boar speimatozoa with Mr 53kD that was later identified as proacrosin (6), the sperm specific protein found within the acrosome. In the present work we show that proacrosin has the capacity to recognize the carbohydrate moiety of a neoglycoprotein probe, BSA-mannose. The evidence suggests that during the early stages of fertilization proacrosin has a dual role, namely to mediate secondary or consolidated binding of spermatozoa to the zona following the acrosome reaction by virtue of its carbohydrate binding activity and after activation to acrosin to help modify the zona so as to facilitate penetration of spermatozoa. PBS-washed boar spermatozoa were,extracted in 0.264M sucrose@nM p-aminobenzamidine (pAB) pH 3.0 for 24hrs at 4OC, centrifuged at 1OOOOgfor 1Omins and soluble proteins separated by SDS-PAGE followed by Western blotting $LO nitrocellulose. Blots were blocked with 3% BSA for 2hr and probed with I-labelled BSA-mannose for a further 2hr. Bound proteins were detected by autoradiography. BSA-mannose was synthesized by the method of (7) and iodinated using Iodogen (8). A polyclonal antito boar sperm proacrosin was prepared and used to probe blotslf$ described previously (6). I-B%- mannose binds strongly to a sperm protein on Fig. la shows that blots with an Mr 53kD that is commensurate with proacrosin as detected by the specific antibody (Fig. lb). Weak binding of the probe was also detected at Mr $$D, 38kD and 18-24kD. This pattern is identical to that found using I-labelled zona glycoproteins from pig eggs (5). The presence of 5m~ pLB had no effect on uptake of the probe. Blots probed with 12’I-BSA (Fig.lc) or pre-i-e serum (Fig.ld) were negative. Fig.le shows the profile of proteins present on blots after staining with Coomassie Blue. These results are similar protein present to the report by Topfer-Petersen et al, (4) of a fucose-binding within the acrosomes of boar spenwitozoa. We identify this carbohydrate binding protein as proacrosin and suggest that it is the ligand molecule that interacts with carbohydrate moieties on zona glycoproteins during the early stages of Proacrosin therefore, may be analogous to the ‘bindin’ protein fertilization. that is found in the acrosome of sea urchin spermatozoa and which is thought to mediate sperm adhesion in Echinoderms (9). Acknowledgements: We are grateful to Davey for synthesis of BSA-mannose. References:

Mr

R.

l.Wassarman PM:Science

235,554(1987) 2.Ahuja KK:ETxp.Cell Res. m,353(1982) 3.Shur BD:Adv. Exp.Med.Biol. m,79(1986) 4.l%pferPetersen E et ZHistochem. 83,139 (1985) 5.Brown CR, Jones R: Developnt39,333(1987) 6.Jones R, Brown CR:Exp.Cell Res?&,in press (1987) 7. Swartz BA, Gray GR:Arch. Biochem.Biophys.l81,542 (1977) 8.Markwell MA, Fox CF:Biochemz 4807 (1978) 9. Vacquier VD, May GW:Proc. Natl.Acad.Sci(USA)E,2456(1977)

Received:

21.9.87

0309-1661/67/110833-01/$03.00/0

Accepted:

2.10.87 @ 1987 Academic

Press Inc. (London)

Ltd.