Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs. Role of the alpha(6) integrin subunit.
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.
Bigler
D
D
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
Takahashi
Y
Y
Chen
M S
MS
Almeida
E A
EA
Osbourne
L
L
White
J M
JM
D051722:ADAM Proteins; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D016367:Baculoviridae; D019483:Disintegrins / Q000378:metabolism*; D005260:Female; D000072196:Fertilins; D039503:Integrin alpha6; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D019483:Disintegrins; D039503:Integrin alpha6; D008562:Membrane Glycoproteins; D051722:ADAM Proteins; C000606458:Adam2 protein, mouse; C000606459:Adam2 protein, rat; D000072196:Fertilins; D008666:Metalloendopeptidases