Identification of key functional amino acids of the mouse fertilin beta (ADAM2) disintegrin loop for cell-cell adhesion during fertilization

Fertilin beta (also known as ADAM2) is a cell adhesion molecule on the surf ace of mammalian sperm that participates in sperm-egg membrane binding. Fer tilin beta is a member of the molecular family known as ADAMs or MDCs. Thes e proteins have a disintegrin domain with homology to integrin ligands foun d in snake venoms; several of these snake proteins have an RGD tripeptide p resented on an extended "disintegrin loop." However, fertilin beta lacks an RGD tripeptide and instead has the consensus sequence X(D/E)ECD (QDECD in mouse fertilin beta) in its putative disintegrin loop, and there is controv ersy over which amino acids comprise the active site of the fertilin beta d isintegrin loop. We have used point-mutated versions of the sequence AQDECD VT and two bioassays to identify the key functional amino acids of this seq uence from the mouse fertilin beta disintegrin domain. Amino acid substitut ions for the terminal aspartic acid residue of the QDECD sequence result in dramatically reduced activities in the two assays for protein function, im plicating the terminal aspartic acid residue as critical for protein functi on. 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 beta disintegrin loop, es pecially the terminal D residue, contributes more to the protein's activity than does the QDE sequence that aligns with the RGD tripeptide in other di sintegrins.