Sequence-specific interaction between the disintegrin domain of mouse ADAM2 (Fertilin beta) and murine eggs - Role of the alpha(6) integrin subunit

Little is set known about the biological and biochemical properties of the disintegrin-like domains of ADAM ((a) under bar (d) under bar isintegrin (a ) under bar nd (m) under bar etalloprotease) proteins. Mouse ADAM 2 2 (mADA M 2; fertilin beta) is a sperm surface protein involved in murine fertiliza tion. We produced recombinant proteins containing the disintegrin-like doma in of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D+C) contained a signal sequence followed by the disi ntegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing th e disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D+C, as well as the D domain of the bacteria l construct (released with thrombin), bound to the microvillar surface of m urine eggs. Using concentrations in the range of 1 to 5 mu m, both recombin ant proteins strongly inhibited sperm-egg binding and fusion; the baculovir us-produced protein exhibited a somewhat greater extent of inhibition (simi lar to 75 versus similar to 55% maximal inhibition). Substitution of alanin e for each of the five charged residues within the disintegrin loop of mADA M 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the functi on blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfuncti on-blocking anti-alpha(6) monoclonal antibody. Binding was also inhibited b y a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2.