Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome

Abstract

Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were M(r) 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2-D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the protein's functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of acrosomal exocytosis.

Sea star tenacity mediated by a protein that fragments, then aggregates

“…S2), precisely comprising the first 3 aa PHY of the fourth fragment's N-terminal peptide. Interestingly, the sequence GDPH has been described as a site of cleavage in several proteins, such as mucins (31,32), the heavy chain 3 of the preα-inhibitor (33), repulsive axon guidance molecules (34,35), and zonadhesin (36). All these proteins are synthesized in the form of precursors which undergo cleavage between the aspartate and proline in the GDPH sequence.…”

The role of the acrosomal matrix in fertilization

“…A the perimeter of the acrosome in intact ejaculated boar spermatozoa while in sperm with disrupted acrosomal membranes, the protein was detected at the leading edge of acrosomal matrix (Bi et al, 2003).…”

“…For example, porcine and human sperm have smaller acrosomes that do not exhibit the prominent domains seen by transmission electron microscopy in guinea pig sperm. By immunoelectron microscopy, zonadhesin localizes to the perimeter of the acrosome in intact ejaculated boar spermatozoa or cauda epididymal hamster sperm, and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes (Bi et al, 2003, Olson et al, 2004. On the other hand, SP-10 is most abundant within the principal segment and posterior bulb of the equatorial segment of the human sperm acrosome (Foster et al, 1994).…”

“…Subsequently, cDNAs or genes have been partially or completely sequenced from mouse, human, rabbit, hamster, and non-human primates (Bi et al, 2003, Gao and Garbers, 1998, Gao et al, 1997, Hardy and Garbers, 1995, Herlyn and Zischler, 2007, Lea et al, 2001, Olson et al, 2004) Testicular porcine zonadhesin is extractable with non-ionic detergents and would not be considered a component of the acrosomal matrix by the operational definition listed above (Bi et al, 2003). In round and elongating spermatids, zonadhesin localizes to the developing outer acrosomal membrane.…”

“…Furthermore, additional studies suggest that some matrix proteins may interact with either the outer or inner acrosomal membrane proteins. As previously mentioned, zonadhesin, which is considered an acrosomal matrix protein in mature boar and hamster sperm is initially synthesized as an acrosomal membrane protein that becomes proteolytically processed and incorporated into the matrix as the spermatid/sperm matures (Bi et al, 2003, Olson et al, 2004. In addition, inner acrosomal membrane proteins such as CD46 (membrane cofactor protein) may also influence the dispersion of acrosomal matrix components during the course of acrosomal exocytosis; males null for CD46 display a higher incidence of spontaneous acrosomal exocytosis and heightened fertilizing ability, suggesting that CD46 may have some role in regulating sperm acrosomal exocytosis (Inoue et al, 2003).…”