Identification of a hyaluronic acid (HA) binding domain in the PH‐20 protein that may function in cell signaling

Abstract The macaque sperm surface protein PH‐20 is a hyaluronidase, but it also interacts with hyaluronic acid (HA) to increase internal calcium ( [Ca 2+ ] i ) in the sperm cell. A region of the PH‐20 molecule, termed Peptide 2 (aa 205–235), has amino acid charge homology with other HA binding proteins. The Peptide 2 sequence was synthesized and two recombinant PH‐20 proteins were developed, one containing the Peptide 2 region (G3, aa 143–510) and one without it (E12, aa 291–510). On Western blots, affinity‐purified anti‐Peptide 2 IgG recognized the 64 kDa band corresponding to PH‐20 in acrosome intact sperm and, under reducing conditions, recognized the whole 67 kDa PH‐20 and the endoproteolyzed N‐terminal fragment of PH‐20. HA conjugated to a photoaffinity substrate specifically bound to sperm surface PH‐20. Indirect immunofluorescence demonstrated that Fab fragments of anti‐Peptide 2 IgG bound to the head of live sperm. Biotinylated HA was bound by Peptide 2 and by sperm extracts in a microplate binding assay, and this binding was inhibited by Fab fragments of anti‐Peptide 2 IgG. Biotinylated HA bound to the G3 protein and this binding was inhibited by anti‐Peptide 2 Fab, but HA did not bind to the E12 protein. Fab fragments of anti‐Peptide 2 IgG inhibited the increase in [Ca 2+ ] i induced in macaque sperm by HA. Our results suggest that the Peptide 2 region of PH‐20 is involved in binding HA, which results in the cell signaling events related to the elevation of [Ca 2+ ] i during sperm penetration of the cumulus. Mol. Reprod. Dev. 60: 542–552, 2001. © 2001 Wiley‐Liss, Inc.