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 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.
Vines
C A
CA
Bodega Marine Laboratory, University of California-Davis, Bodega Bay, California, USA.
Li
M W
MW
Deng
X
X
Yudin
A I
AI
Cherr
G N
GN
Overstreet
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D002457:Cell Extracts; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase; D015151:Immunoblotting; D008251:Macaca; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000166:cytology / Q000276:immunology / Q000378:metabolism
D015815:Cell Adhesion Molecules; D002457:Cell Extracts; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20; D002118:Calcium