Characterization of the active site of monkey sperm hyaluronidase

The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides re presenting regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the su rface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molec ule. Western blots of acrosome-reacted spermatozoa showed that, under reduc ing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also label s the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Pep tide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane, fused acrosomal vesicles, acrosomal matrix and inner acrosomal membrane. Fab fragments of anti-peptide 1 IgG, but not anti-pept ide 3 IgG, inhibited hyaluronidase activity of PH-20 from the sperm surface and from extracts of acrosome-reacted spermatozoa at pH 7.0. Fab fragments of both anti-peptide IgGs inhibited sperm hyaluronidase activity at pH 5.0 . It is concluded that the region of PH-20 encompassed by the amino acid se quence 142-172 is essential for hyaluronidase activity at neutral pH, where as the region of amino acid sequence 277-297 may be more important at a low er pH. It is likely that these two regions are the acid/base catalyst site and the nucleophilic site, respectively, of PH-20 hyaluronidases.