Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Abstract

Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.

Figure 1

(A) Comparison of chemically determined amino acid sequences of gp69 and gp64 starting from three different sites in the polypeptide: site 1, N terminus of intact, mature gp69 and gp64; site 2, N terminus of the proteolytically processed forms of gp69 and gp64 (gp66 and gp61) after egg activation; and site 3, the 65- and 60-kDa forms generated by treatment of eggs with type I collagenase. (B) Deglycosylation of gp69 and gp64. Intact gp69/64, PNGase F-treated, and trifluoromethanesulfonic acid-treated gp69/64 were separated by using SDS/PAGE, transferred onto a membrane, and Western blotted with the specific anti-gp69/64 antibody.

Figure 2

cDNA sequence and translated single-letter amino acid sequence of gp69/64 protein. The N-terminal pre-pro-peptide sequence and the C-terminal peptide sequence after the putative furin cleavage site (RRKR) is italicized. The chemically determined peptide sequences are underlined and in boldface. The four putative N-glycosylation sites are underlined. The polyadenylation signal (AATAAA) is in boldface.

Figure 3

Protein homology of Xenopus gp69/64 VE protein with mammalian ZP2. (A) The sequence of Xenopus gp69/64 was compared with the sequence of ZP2 from human, mouse, and pig (–22). At each position, residues that are identical in 3 of 4 sequences are boxed. The ZP domain is overlined. The 16 conserved cysteines are indicated by closed squares. The putative furin-cleavage site is underlined. (B) Comparison of hydropathy plots of Xenopus gp69/64 and mouse and human ZP2s. Hydropathy plots were made by using DNA Strider and the Kyte–Doolittle algorithm. Plots were aligned at the conserved ZP domain. A putative signal peptide (SP) and a C-terminal hydrophobic domain (TM) are present in all three sequences. (C) Protein-matrix plots (pam250 matrix) of mouse ZP2 protein vs. the Xenopus gp69/64. Parameters: Window size = 8, Minimum % score = 60; Hash value = 2. (D) Western blot of total VE proteins with polyclonal antiserum against mouse ZP2 protein.

Figure 4

The N terminus of gp69/64 is essential for sperm binding. (A) Comparison of the inhibitory activity of gp69/64, gp66/61, and gp65/60 on sperm–egg binding. Each protein (5 μg/ml) was used as competitor in the sperm-binding competition assays. (B) Comparison of anti-gp69/64 antibody staining of the surface of a dejellied unfertilized egg before (Left) and after (Right) treatment with type I collagenase. The apparent “staining” seen after collagenase on the vegetal hemisphere is an artifact caused by autofluorescence.