LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm: evidence that the antigen recognized is not...

In the mouse, a 95 kD sperm protein has been identified as a putative receptor for the zona pellucida glycoprotein ZP3. The 95 kD sperm protein is a tyrosine kinase substrate, with phosphorylation on tyrosine stimulated upon zona pellucida binding. The latter finding is observed not only in live cells but also in isolated sperm membranes and in an electroeluted 95 kD protein. Stimulation of 95 kD protein tyrosine phosphorylation by zona pellucida is completely abolished by tyrosine kinase inhibitors, which effectively inhibit the sperm acrosome reaction. Since receptor oligomerization by ZP3 is essential for acrosome reaction triggering, we hypothesized that application of an external crosslinking agent will lead to the acrosome reaction, even in the absence of natural ligand ZP3. Here, we report the generation of a mouse monoclonal antibody (mAb) raised against the 95 kD protein. This antibody, termed LL95, mimics the bioactivities of ZP3 in inhibiting sperm-zona binding and inducing the acrosome reaction. The latter depends on receptor oligomerization. Immunolocalization revealed that the LL95 antigen is restricted to the head surface in the acrosomal region of live sperm. Thus, LL95 fulfills several criteria predicted for an antibody that recognizes a sperm receptor for the zona pellucida. Recently, it was reported that the amino acid sequence of the 95 kD protein we described corresponds to a mouse hepatoma hexokinase (Kalab et al., 1994: J Biol Chem 269:3810-3817). Although both hexokinase and LL95 antigen migrate at 95 kD in nonreducing gels, we show here that LL95 does not recognize hexokinase. Identification of different proteins is clear where hexokinase is a 116 kD protein and LL95 recognizes sperm proteins of 110 and 130 kD. Moreover, mAb anti-phosphotyrosine immunoprecipitates LL95 antigen under conditions where hexokinase is absent. Use of anti-hexokinase antibodies in gamete interaction assays failed to demonstrate any effect on either sperm-zona binding or acrosome reaction triggering. Finally, antihexokinase antibodies bind to a sperm tail antigen, thus direct involvement of hexokinase in gamete interaction seems improbable.