Binding of recombinant human proacrosin/acrosin to zona pellucida (ZP) glycoproteins. I. Studies with recombinant human ZPA, ZPB, and ZPC  Laura I. Furlong, Ph.D., Jeffrey D. Harris, Ph.D., Mónica H. Vazquez-Levin, Ph.D.  Fertility and Sterility  Volume 83, Issue 6, Pages 1780-1790 (June 2005) DOI: 10.1016/j.fertnstert.2004.12.042 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Acrosin recombinant proteins. (A) Graphical representation for the primary structure of native preproacrosin (adapted from Baba et al. [57]) and of recombinant proacrosin and N-terminal expression fragments. Domain I (DI) and Domain II (DII) have sequence similarities with trypsin-like proteases, while Domain III (DIII) is unique to proacrosin. Recombinant proteins from human proacrosin Rec-40, Rec-30, Rec-20, and Rec-10 were obtained from expression of a full-length cDNA construct (42), while Rec-6 was obtained by expression of a cDNA fragment encoding residues 1–59 of human proacrosin. Native preproacrosin is shown for comparison. (B) Purified bacterial recombinant proteins of acrosin (Rec-40, Rec-30, Rec-20, Rec-10, and Rec-6) were analyzed by 12% SDS-PAGE under reducing conditions followed by CBB staining. MWM = molecular weight markers in kDa. Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Binding of recombinant acrosin (Rec-30) to individual rec-hZP glycoproteins. Ten pmol of Rec-30 were immobilized onto microtiter plates and incubated with rec-hZPA, rec-hZPB, and rec-hZPC (330 nM) as described in Materials and Methods. (a) Result different from rec-hZPA binding value (ANOVA, Tukey HSD test for post hoc comparisons, P<.0005; n = 4). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 (A) Effect of rec-hZP heat-denaturating treatment upon binding to Rec-30. Ten pmol of Rec-30 were incubated with each rec-hZP glycoprotein in native (ZPA, ZPB, ZPC) and heat-denaturing conditions (DZPA, DZPB, DZPC). Proteins were incubated at 100°C during 10 minutes for heat denaturation, and the assay was performed as described. Native vs. heat-denaturated glycoprotein binding activity was compared using the Student’s t-test (n = 3). (a) P<.0005. (b) P<.05. (B) The effect of dextran sulfate upon binding of Rec-30 to heated rec-hZPA. The experimental conditions were the same as described previously, with the addition of 50 μM of dextran sulfate. Binding activity in the absence vs. in the presence of dextran sulfate was compared using the Student’s t-test (a, b: P<0.01; n = 3). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Binding of Rec-30 to rec-hZPA in the presence dextran sulphate. Rec-30 (10 pmol) was incubated with 330 nM of rec-hZPA in the absence or presence of 10 μM and 50 μM of dextran sulphate. Results were compared by ANOVA and Tukey HSD test. Statistical significance is indicated (a: P<.001; b: P<.0005; n = 3). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Binding of Rec-30 to rec-hZPA in the presence of BSA-fucose (Fuc) and BSA-glucose (Glc). Rec-30 (10 pmol) was incubated with 330 nM of rec-hZPA with no additives (buffer) or in the presence of 1.5 μM and 3 μM of BSA-Fuc or BSA-Glc. Results were compared by ANOVA and Tukey HSD test; statistical differences are indicated (a: P<.005; b: P<.05; n = 4). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 6 Binding of Rec-30 to rec-hZPA in the presence of mannose, glucose, and N-acetylglucosamine. Rec-30 (10 pmol) was incubated with 330 nM of rec-hZPA with no additive (buffer) or in the presence of mannose (Man), glucose (Glc), or N-acetylglucosamine (GlcNAc); sugar concentrations are indicated in the figure. Results were compared by ANOVA and Tukey HSD test. Differences are indicated (a: P<.0005, c: P<.05, d: P<.01; b: difference from result obtained with 67 mM of mannose, P<.0005; n = 3). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 7 Binding of recombinant proacrosin and N-terminal products to rec-hZPA (A), rec-hZPB (B), and rec-hZPC (C). Ten pmol of each acrosin protein were incubated with each ZP glycoprotein at saturation, and binding was analyzed as described in Materials and Methods. Statistical differences were determined by ANOVA and Tukey HSD test: Differences are indicated (a: P<.0005, b: P<.05; N.D. = not detectable; n = 4). Furlong. Human acrosin binding to 2P glycoproteins. Fertil Steril 2005. Fertility and Sterility 2005 83, 1780-1790DOI: (10.1016/j.fertnstert.2004.12.042) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions