Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa.
The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3. The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure. We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein. The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium. After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa. The binding site migrated from the acrosome to the midpiece of the spermatozoa. Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values. The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP.
Tsubamoto
H
H
Department of Obstetrics and Gynecology, Institute of Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, 663-8501, Japan.
Hasegawa
A
A
Nakata
Y
Y
Naito
S
S
Yamasaki
N
N
Koyama
K
K
D000177:Acrosome / Q000378:metabolism; D020101:Acrosome Reaction; D000818:Animals; D001665:Binding Sites; D004527:Egg Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D011817:Rabbits; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606514:ZP1 protein, human; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606519:Zp1 protein, mouse; C000606520:Zp2 protein, mouse; C000606521:Zp3 protein, mouse