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A role for the disintegrin domain of cyritestin, a sperm surface protein belonging to the ADAM family, in mouse sperm-egg plasma membrane adhesion and fusion.

Yuan R, Primakoff P, Myles DG - J. Cell Biol. (1997)

Bottom Line: This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain.Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5.Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, University of California, Davis 95616, USA.

ABSTRACT
Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm-egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (alpha and beta subunits). Fertilin alpha and beta are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin beta functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin beta, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80-90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin beta active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin beta also strongly inhibited (80-90%) both sperm-egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin beta showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin beta in sperm-egg plasma membrane adhesion leading to fusion.

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Sequence alignment of disintegrin domain  active sites of snake disintegrins and guinea pig fertilin β  with mouse ADAM family  members. The sequences  shown are the 13 amino acids  that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin,  and the corresponding 14 amino acids of guinea pig fertilin β and  mouse ADAM family proteins. Italicized residues are those  which align with RGD, and the underlined sequences are the  peptide sequences tested in the adhesion and fusion assay.
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Figure 1: Sequence alignment of disintegrin domain active sites of snake disintegrins and guinea pig fertilin β with mouse ADAM family members. The sequences shown are the 13 amino acids that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin, and the corresponding 14 amino acids of guinea pig fertilin β and mouse ADAM family proteins. Italicized residues are those which align with RGD, and the underlined sequences are the peptide sequences tested in the adhesion and fusion assay.

Mentions: Peptides with sequences from the predicted active sites of the disintegrin domains of ADAM proteins were synthesized by the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, CT). The sequences of the peptides were chosen by sequence alignment with the known active sites of snake disintegrins (RGD) and guinea pig fertilin β (TDE; Fig. 1). The peptides synthesized were each linear, eight residue peptides from mouse fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. Their sequences are underlined in Fig. 1. All of the peptides were purified by HPLC, and their sequences were confirmed by mass spectroscopy.


A role for the disintegrin domain of cyritestin, a sperm surface protein belonging to the ADAM family, in mouse sperm-egg plasma membrane adhesion and fusion.

Yuan R, Primakoff P, Myles DG - J. Cell Biol. (1997)

Sequence alignment of disintegrin domain  active sites of snake disintegrins and guinea pig fertilin β  with mouse ADAM family  members. The sequences  shown are the 13 amino acids  that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin,  and the corresponding 14 amino acids of guinea pig fertilin β and  mouse ADAM family proteins. Italicized residues are those  which align with RGD, and the underlined sequences are the  peptide sequences tested in the adhesion and fusion assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139869&req=5

Figure 1: Sequence alignment of disintegrin domain active sites of snake disintegrins and guinea pig fertilin β with mouse ADAM family members. The sequences shown are the 13 amino acids that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin, and the corresponding 14 amino acids of guinea pig fertilin β and mouse ADAM family proteins. Italicized residues are those which align with RGD, and the underlined sequences are the peptide sequences tested in the adhesion and fusion assay.
Mentions: Peptides with sequences from the predicted active sites of the disintegrin domains of ADAM proteins were synthesized by the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, CT). The sequences of the peptides were chosen by sequence alignment with the known active sites of snake disintegrins (RGD) and guinea pig fertilin β (TDE; Fig. 1). The peptides synthesized were each linear, eight residue peptides from mouse fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. Their sequences are underlined in Fig. 1. All of the peptides were purified by HPLC, and their sequences were confirmed by mass spectroscopy.

Bottom Line: This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain.Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5.Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, University of California, Davis 95616, USA.

ABSTRACT
Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm-egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (alpha and beta subunits). Fertilin alpha and beta are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin beta functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin beta, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80-90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin beta active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin beta also strongly inhibited (80-90%) both sperm-egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin beta showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin beta in sperm-egg plasma membrane adhesion leading to fusion.

Show MeSH
Related in: MedlinePlus