Abstract
Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin beta) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain 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 disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the 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 bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 microM, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (approximately 75 versus approximately 55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfunction-blocking anti-alpha(6) monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2.
Highlights
Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM proteins
We previously provided evidence that the disintegrin-like domain of Mouse ADAM 2 (mADAM 2) interacts with the integrin ␣61: (i) Sperm binding to eggs is inhibited by a function-blocking anti-␣6 monoclonal antibody as well as by a peptide analogue of the disintegrin loop of mouse ADAM (mADAM) 2. (ii) Sperm bind more avidly to ␣6-transfected cells than to their mock-transfected counterparts as well as to cells expressing versus those engineered not to express the 1 integrin subunit. (iii) Sperm binding to somatic cells expressing ␣61 is inhibited by the function-blocking anti-␣6 mAb, GoH3, as well as a disintegrin loop peptide [4]
Expression of Disintegrin-like Domain-containing Constructs of Mouse ADAM 2 in Insect and Bacterial Cells—Our goal was to generate a recombinant protein to test the biological activity of the disintegrin-like domain of mADAM 2
Summary
Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-␣6 monoclonal antibody, GoH3, but not by a nonfunctionblocking anti-␣6 monoclonal antibody. A recombinant protein produced in insect cells containing the disintegrin-like and cysteine-rich domains of atrolysin A inhibits ADP- and collagen-induced platelet aggregation with similar IC50 values of ϳ0.3 and ϳ0.47 M, respectively. What is known can be summarized as follows: Peptide analogues of the disintegrin loops of several sperm ADAMs inhibit sperm-egg binding and fusion using peptide concentrations of 100 –500 M (4 –13). This paper is available on line at http://www.jbc.org construct inhibited sperm-egg binding and fusion by ϳ50 and ϳ25%, respectively [18]
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