Biochemical Characterization of Two Major Proteins of the Hamster Sperm Acrosomal Matrix

An acrosomal fraction, termed the ALM (acrosomal lamina complex) was isolated from hamster cauda epididymal spermatozoa that contain specific domains of the acrosomal matrix and an adherent detergent-insoluble complex, termed the acrosomal lamina, which is derived from the outer acrosomal membrane. By SDS-PAGE, the ALM fraction exhibited two major polypeptides of Mr=29,000 (ALM29) and Mr=22,000 (ALM22). We have also shown that an ALM complex binds both acrosin and N-acetylglucosaminidase (NAGA) in a dose-dependent manner and acrosin binds to the ALM29 polypeptide only. The objective of the present study is to investigate the binding efficiency of acrosin to the V8 protease-generated peptides of ALM29 and ALM22 polypeptides, to examine the presence of phosphate groups on both ALM29 and ALM22 polypeptides and to identify the binding competency of dephosphorylated ALM29 polypeptide to acrosin. Diagonal gel electrophoresis was performed to investigate whether both ALM29 and ALM22 polypeptides are joined by disulfide bridge(s). Both polypeptides migrate on a diagonal line with a comparable Rf in both non-reduced and reduced conditions suggesting that both polypeptides are not joined by disulfide bridge(s). All V8 protease-digested peptides of ALM29 and ALM22 showed immunoreactive bands when stained with anti-ALM22 antibody suggesting that all peptides are antigenically related family. Both ALM29 and ALM22 polypeptides were stripped out of the ALM complex by high pH (pH-11) extraction and were treated with alkaline phosphatase followed by immunoblot analysis. Both ALM29 and ALM22 polypeptides showed a reduction in size (~3 kDa) by alkaline phosphatase treatment. A sedimentation assay was employed to determine whether alkanine phosphate treated ALM29 polypeptide possesses acrosin binding. Dephosphorylated ALM29 polypeptide revealed a significant reduction (~50%) in acrosin binding in comparison with acrosin to a native ALM29 polypeptide. Our studies conclude that both ALM29 and ALM22 polypeptides are phosphorylated and demonstrate the role of phosphate group of ALM29 polypeptide in acrosin binding.