Analysis of Fertilin α (ADAM1)-mediated Sperm-Egg Cell Adhesion during Fertilization and Identification of an Adhesion-mediating Sequence in the Disintegrin-like Domain

Abstract

Fertilin ␣ (also known as ADAM1) is a member of the ADAM (A disintegrin and A metalloprotease domain) family of proteins. In this study, we examine the mechanism of mouse fertilin ␣'s in adhesion of sperm to the egg plasma membrane during fertilization. We find that recombinant forms of fertilin ␣ corresponding to either the disintegrin-like domain or the cysteine-rich domain and the EGF-like repeat can perturb sperm-egg binding, suggesting that both of these domains can participate in fertilin ␣-mediated adhesion events. In further examination of the fertilin ␣ disintegrin-like domain, we find that a subdomain of disintegrin-like domain with the sequence DLEECDCG outside the putative disintegrin loop but with homology to the fertilin ␤ disintegrin loop can inhibit the binding of both sperm and recombinant fertilin ␣ to eggs, suggesting that this is an adhesionmediating motif of the fertilin ␣ disintegrin-like domain. This sequence also inhibits the binding of recombinant fertilin ␤ to eggs and thus is the first peptide sequence found to block two different sperm ligands. Finally, a monoclonal antibody to the tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant fertilin ␣ to eggs in one type of binding assay, suggesting that, under certain conditions, fertilin ␣ may interact with a KMC.8-sensitive binding site on the egg plasma membrane. ADAM1 (for A disintegrin and A metalloprotease) proteins comprise a recently identified and rapidly growing molecular family of membrane proteins. To date, ϳ30 ADAMs have been identified in a variety of animal species, including several mammals, Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans (1, 2). The conserved domain structure of ADAMs (see Fig. 1A) and functional analyses of these proteins indicate that members of this family have functions as proteases and/or as cell adhesion molecules. In the ADAMs that have been described to function as cell adhesion molecules, the adhesive activity generally appears to be attributable to the disintegrin-like domain, a domain with homology to integrin ligand-like snake venom polypeptides. These venom polypeptides, known as disintegrins, contain an RGD tripeptide, presented at the end of an extended loop structure called the "disintegrin loop" (3). Although ADAMs share significant sequence homology in their disintegrin-like domains to snake venom disintegrins, they do not have RGD sequences in their putative disintegrin loops (with the exception of human ADAM15). Various studies have shown that the adhesion-mediating sequence of several ADAMs appears to be a short sequence within the putative disintegrin loop, such as the ECDcontaining sequences in fertilin ␤ (4, 5), ADAM9 (6), and ADAM23 (7), or the QCD-containing sequence in cyritestin (8).Among the cell adhesion events that are mediated by ADAMs are the interactions between mammalian gamete plasma membranes during fertilization. On mouse sperm, there are at least three ADAMs that appear to participate in sperm-egg binding: fertilin ␣ (ADAM1), fertili...

The mechanism of sperm–oocyte fusion in mammals

“…In constrast, CD9 was reported to play a role in sperm-oocyte binding. Zona pellucida-free oocytes treated with anti-CD9 antibodies showed reduced levels of binding to the ligands; fertilin a, fertilin b and cyritestin on sperm when the antibodies were immobilized on small beads , Takahashi et al 2001, Wong et al 2001, Zhu & Evans 2002. Although the functional difference between the lack of CD9 and treatment with anti-CD9 antibodies is not clear, the antibodies may disturb the strength of adhesions mediated by a molecule such as a6 integrin involved in the multi-protein complex together with CD9.…”

Localization of CD9 in pig oocytes and its effects on sperm–egg interaction

“…Further evidence has indicated that sperm were able to adhere to the plasma membrane of zona pellucida (ZP)-free oocytes from CD9 knockout mice; however, very few sperm could fuse with the oocyte membrane (Le Naour et al 2000, Miyado et al 2000. Reduced sperm binding was also found in ZP-free mouse oocytes treated with anti-CD9 monoclonal antibodies , Takhashi et al 2001, Wong et al 2001, Zhu & Evans 2002. Sperm -oocyte fusion was also decreased when the oocytes treated with anti-CD9 antibody were inseminated (Miller et al 2000).…”

Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel α9β1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases

“…2B demonstrate that the toxin encoding ESTs are dominated by SVMPs (58%) and bradykinin-potentiating peptides (18%) relative to PLA 2 s and CTLs (both 9%) with lower (1-2%) relative expression levels of serine proteinases, growth factors and L-amino oxidases. Examination of the GOannotated proteolysis and peptidolysis category also identified two clusters of novel aspartic proteases representing 2% of a Position analogous to IB-I (α 2 β 1 ) ECD-like motif (Kamiguti et al, 1997;Zhu et al, 2000;Wong et al, 2001). b Conserved IB-Ib (α 9 β 1 ) motif in ADAMS (Eto et al, 2000).…”