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Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

Choi H, Jin S, Kwon JT, Kim J, Jeong J, Kim J, Jeon S, Park ZY, Jung KJ, Park K, Cho C - PLoS ONE (2016)

Bottom Line: Monkey ADAM2 was found to associate with chaperone proteins in testis.In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm.Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

ABSTRACT
The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

No MeSH data available.


Related in: MedlinePlus

Coimmunoprecipitation of ADAM2 in mouse testes.A and B. Testes from wild-type and Adam2-/- mice were lysed with 1% NP-40 buffer, the tissue lysates were immunoprecipitated with A. anti-mADAM2-CyT-1 and B. anti-mADAM2-CyT-2, and the resolved immunoprecipitates were immunoblotted with anti-mADAM2-D. Normal rabbit serum was used as a control. C and D. Lysates were precipitated with C. anti-mADAM2-Cyt-1 and D. anti-mADAM2-CyT-2 and analyzed by immunoblotting with anti-mADAM2-D, anti-mADAM1B, and anti-mADAM3. Experiments were repeated five times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviations: WT, wild-type; A2-/-, Adam2-/; TL, tissue lysate (100 μg); IP, immunoprecipitated protein (1 mg); NRS, normal rabbit serum; and IB, immunoblotting.
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pone.0158321.g002: Coimmunoprecipitation of ADAM2 in mouse testes.A and B. Testes from wild-type and Adam2-/- mice were lysed with 1% NP-40 buffer, the tissue lysates were immunoprecipitated with A. anti-mADAM2-CyT-1 and B. anti-mADAM2-CyT-2, and the resolved immunoprecipitates were immunoblotted with anti-mADAM2-D. Normal rabbit serum was used as a control. C and D. Lysates were precipitated with C. anti-mADAM2-Cyt-1 and D. anti-mADAM2-CyT-2 and analyzed by immunoblotting with anti-mADAM2-D, anti-mADAM1B, and anti-mADAM3. Experiments were repeated five times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviations: WT, wild-type; A2-/-, Adam2-/; TL, tissue lysate (100 μg); IP, immunoprecipitated protein (1 mg); NRS, normal rabbit serum; and IB, immunoblotting.

Mentions: ADAM2 is known to assemble into complexes with ADAM1A, ADAM1B, ADAM3, ADAM6 and potentially other ADAMs [15, 17–19]. In an effort to further catalog the ADAM2-associated proteins, we performed coimmunoprecipitation analyses using the anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 antibodies in mouse testis. Both antibodies were able to effectively precipitate the 100-kDa precursor form of ADAM2 in testis (Fig 2A and 2B). WT testicular protein samples immunoprecipitated with anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 were immunoblotted with antibodies against ADAM1B, ADAM2-disintegrin, and ADAM3 (no antibody against ADAM1A was available). Testicular protein samples from Adam2-/- mice were analyzed as a control. Consistent with a previous report [19], WT testicular protein samples immunoprecipitated with the ADAM2 antibodies contained ADAM2 and ADAM3 (Fig 2C and 2D). The ratio of precipitated protein to the total (input) appeared to be lower for ADAM3 than for ADAM2, suggesting that ADAM2-free ADAM3 may be present. We also observed ADAM1B coimmunoprecipitated with ADAM2 when anti-mADAM2-CyT-1 was used (Fig 2C). However, we failed to identify ADAM1B in proteins immunoprecipitated with anti-mADAM2-CyT-2 (Fig 2D). This indicates that the portion of ADAM2 recognized by anti-mADAM2-CyT-2 does not associate with ADAM1B, further suggesting that differential ADAM2 complex formation in the mouse testis is governed (at least in part) by the cytoplasmic tail domain. Alternatively, it is possible that anti-mADAM2-CyT-2 antibody binds the cytoplasmic tail of ADAM2 in the manner which prevents/destabilizes the interaction with ADAM1B.


Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

Choi H, Jin S, Kwon JT, Kim J, Jeong J, Kim J, Jeon S, Park ZY, Jung KJ, Park K, Cho C - PLoS ONE (2016)

Coimmunoprecipitation of ADAM2 in mouse testes.A and B. Testes from wild-type and Adam2-/- mice were lysed with 1% NP-40 buffer, the tissue lysates were immunoprecipitated with A. anti-mADAM2-CyT-1 and B. anti-mADAM2-CyT-2, and the resolved immunoprecipitates were immunoblotted with anti-mADAM2-D. Normal rabbit serum was used as a control. C and D. Lysates were precipitated with C. anti-mADAM2-Cyt-1 and D. anti-mADAM2-CyT-2 and analyzed by immunoblotting with anti-mADAM2-D, anti-mADAM1B, and anti-mADAM3. Experiments were repeated five times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviations: WT, wild-type; A2-/-, Adam2-/; TL, tissue lysate (100 μg); IP, immunoprecipitated protein (1 mg); NRS, normal rabbit serum; and IB, immunoblotting.
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Related In: Results  -  Collection

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pone.0158321.g002: Coimmunoprecipitation of ADAM2 in mouse testes.A and B. Testes from wild-type and Adam2-/- mice were lysed with 1% NP-40 buffer, the tissue lysates were immunoprecipitated with A. anti-mADAM2-CyT-1 and B. anti-mADAM2-CyT-2, and the resolved immunoprecipitates were immunoblotted with anti-mADAM2-D. Normal rabbit serum was used as a control. C and D. Lysates were precipitated with C. anti-mADAM2-Cyt-1 and D. anti-mADAM2-CyT-2 and analyzed by immunoblotting with anti-mADAM2-D, anti-mADAM1B, and anti-mADAM3. Experiments were repeated five times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviations: WT, wild-type; A2-/-, Adam2-/; TL, tissue lysate (100 μg); IP, immunoprecipitated protein (1 mg); NRS, normal rabbit serum; and IB, immunoblotting.
Mentions: ADAM2 is known to assemble into complexes with ADAM1A, ADAM1B, ADAM3, ADAM6 and potentially other ADAMs [15, 17–19]. In an effort to further catalog the ADAM2-associated proteins, we performed coimmunoprecipitation analyses using the anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 antibodies in mouse testis. Both antibodies were able to effectively precipitate the 100-kDa precursor form of ADAM2 in testis (Fig 2A and 2B). WT testicular protein samples immunoprecipitated with anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 were immunoblotted with antibodies against ADAM1B, ADAM2-disintegrin, and ADAM3 (no antibody against ADAM1A was available). Testicular protein samples from Adam2-/- mice were analyzed as a control. Consistent with a previous report [19], WT testicular protein samples immunoprecipitated with the ADAM2 antibodies contained ADAM2 and ADAM3 (Fig 2C and 2D). The ratio of precipitated protein to the total (input) appeared to be lower for ADAM3 than for ADAM2, suggesting that ADAM2-free ADAM3 may be present. We also observed ADAM1B coimmunoprecipitated with ADAM2 when anti-mADAM2-CyT-1 was used (Fig 2C). However, we failed to identify ADAM1B in proteins immunoprecipitated with anti-mADAM2-CyT-2 (Fig 2D). This indicates that the portion of ADAM2 recognized by anti-mADAM2-CyT-2 does not associate with ADAM1B, further suggesting that differential ADAM2 complex formation in the mouse testis is governed (at least in part) by the cytoplasmic tail domain. Alternatively, it is possible that anti-mADAM2-CyT-2 antibody binds the cytoplasmic tail of ADAM2 in the manner which prevents/destabilizes the interaction with ADAM1B.

Bottom Line: Monkey ADAM2 was found to associate with chaperone proteins in testis.In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm.Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

ABSTRACT
The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

No MeSH data available.


Related in: MedlinePlus