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

The macaque sperm surface protein PH-20 is a hyaluroniclase, but it also in teracts with hyaluronic acid (HA) to increase internal calcium ([Ca2+](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 P eptide 2 sequence was synthesized and two recombinant PH-20 proteins were d eveloped, one containing the Peptide 2 region (G3, aa 143-510) and one with out 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 photo affinity substrate specifically bound to sperm surface PH-20. Indirect immu nofluorescence 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 sp erm 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 pro tein and this binding was inhibited by anti-Peptide 2 Fab, but HA did not b ind to the E12 protein. Fab fragments of anti-Peptide 2 IgG inhibited the i ncrease in [Ca2+](i) induced in macaque sperm by HA. Our results suggest th at the Peptide 2 region of PH-20 is involved in binding HA, which results i n the cell signaling events related to the elevation of [Ca2+](i) during sp erm penetration of the cumulus. Mol. Reprod. Dev. 60: 542-552, 2001. (C) 20 01 Wiley-Liss, Inc.