Identification of Key Functional Amino Acids of the Mouse Fertilin β (ADAM2) Disintegrin Loop for Cell-Cell Adhesion during Fertilization

Abstract

Fertilin ␤ (also known as ADAM2) is a cell adhesion molecule on the surface of mammalian sperm that participates in sperm-egg membrane binding. Fertilin ␤ is a member of the molecular family known as ADAMs or MDCs. These proteins have a disintegrin domain with homology to integrin ligands found in snake venoms; several of these snake proteins have an RGD tripeptide presented on an extended "disintegrin loop." However, fertilin ␤ lacks an RGD tripeptide and instead has the consensus sequence X(D/E)ECD (QDECD in mouse fertilin ␤) in its putative disintegrin loop, and there is controversy over which amino acids comprise the active site of the fertilin ␤ disintegrin loop. We have used point-mutated versions of the sequence AQDECDVT and two bioassays to identify the key functional amino acids of this sequence from the mouse fertilin ␤ disintegrin domain. Amino acid substitutions for the terminal aspartic acid residue of the QDECD sequence result in dramatically reduced activities in the two assays for protein function, implicating the terminal aspartic acid residue as critical for protein function. Substitutions for the glutamic acid and the cysteine residues in the QD-ECD sequence result in slight reductions in activity, whereas substitution of the first aspartic acid has virtually no effect. These data suggest that the conserved ECD sequence of the mouse fertilin ␤ disintegrin loop, especially the terminal D residue, contributes more to the protein's activity than does the QDE sequence that aligns with the RGD tripeptide in other disintegrins.The interactions of gamete plasma membranes are mediated by multiple cell adhesion molecules on the surfaces of the sperm and egg. One of these cell adhesion molecules is the mammalian sperm protein fertilin ␤ (previously known as PH-30; also known as ADAM2). The involvement of fertilin ␤ in sperm-egg adhesion during fertilization is well established based on studies with synthetic peptides, antibodies, recombinant proteins, and the subfertile phenotype of male fertilin ␤ knock-out mice (summarized in Ref. 1). Of particular interest is the domain of fertilin ␤ that has homology to a family of integrin ligands originally identified in snake venoms. This domain of fertilin ␤, known as the disintegrin domain, has been implicated in the interactions of fertilin ␤ with the egg membrane in a variety of species (2-9).A large number of disintegrin domain-containing proteins have been identified. These include members of the molecular family known as ADAMs (for A disintegrin and A metalloprotease) or MDCs (for metalloprotease/disintegrin/cysteine-rich) (10, 11), of which fertilin ␤ is a member. Disintegrin domains are also present in a number of snake venom proteins, which are called simply disintegrins, reprolysins, or snake venom metalloproteases. These snake venom polypeptides range in size from small peptide chains of Ͻ50 amino acids to multidomain proteins of several hundred amino acids that are similar in domain structure to ADAM proteins, having a metalloprotease domain and a cy...

The Lymphocyte Metalloprotease MDC-L (ADAM 28) Is a Ligand for the Integrin α4β1

“…The Putative Disintegrin Loop Is Not Required for ␣ 4 ␤ 1 Binding-Previous studies investigating the interaction of snake venom disintegrin peptides and disintegrin-like domains of ADAMs have used peptides to localize the integrin recognition site to a region designated as the "disintegrin loop" (21)(22)(23). A synthetic peptide (KGGVCRPAKDECDL) based on the putative disintegrin loop of MDC-L did not inhibit adhesion of Jurkats to rDis-Fc at 1 mg/ml (data not shown).…”

“…The disintegrin-like domains of ADAMs 1-3 expressed on the surface of sperm interact with the integrin ␣ 6 ␤ 1 in association with CD9 and CD98 on the egg surface (18 -23). Recognition of ADAMs 2 and 3 by ␣ 6 ␤ 1 requires the residues DECD located within a region of the disintegrin domain, designated the disintegrin loop, that corresponds to an extended loop in the snake venom peptides that typically contains an RGD sequence required for integrin binding (21)(22)(23). Human ADAM 15 contains an RGD sequence within the disintegrin loop region and is recognized by the integrins ␣ v ␤ 3 and ␣ 5 ␤ 1 (24 -26).…”

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

“…Bacterial Alkaline Phosphatase-presented Peptides-Bacterial alkaline phosphatase (BAP)-presented peptides were generated as described previously (4). Oligonucleotide sequences that were used are shown in Table I.…”

“…Fusion proteins were expressed and purified on a nickel column as described previously (4). Egg Collection, Zona Pellucida Removal, and IVF-Metaphase IIarrested eggs were obtained from superovulated 6 -9-week-old CF-1 mice (Harlan, Indianapolis, IN); egg collection and cumulus cell removal were performed as described previously (4). All gamete cultures were performed in Whitten's medium (20) containing 22 mM NaHCO 3 and 15 mg/ml bovine serum albumin (Albumax I; Life Technologies, Inc.) at 37°C in 5% CO 2 in air.…”

“…9). As noted above, fertilin ␤ and cyritestin, as well as several other ADAMs, appear to mediate cell adhesion through short peptide sequences within the putative disintegrin loop of the disintegrinlike domain (4,5,8). With regard to fertilin ␣, studies of the putative disintegrin loop have been inconclusive, with a synthetic peptide corresponding to the fertilin ␣ disintegrin loop (AEDVCDLP) having a modest inhibitory effect on sperm-egg interactions but with the scrambled control peptide (PDCE-VADL) also having some effect (8).…”