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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

Immunoprecipitation with anti-hADAM2-CyT.Immunoprecipitation of ADAM2 was carried out using monkey testis lysates. Immunoprecipitation using normal rabbit serum was performed as a negative control. Immunoprecipitated lysates were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gel. Abbreviations: TL, tissue lysate (100 μg); S, supernatant; and IP, immunoprecipitated protein (1 mg); and NRS, normal rabbit serum.
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pone.0158321.g008: Immunoprecipitation with anti-hADAM2-CyT.Immunoprecipitation of ADAM2 was carried out using monkey testis lysates. Immunoprecipitation using normal rabbit serum was performed as a negative control. Immunoprecipitated lysates were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gel. Abbreviations: TL, tissue lysate (100 μg); S, supernatant; and IP, immunoprecipitated protein (1 mg); and NRS, normal rabbit serum.

Mentions: In the course of analyzing the immunoreactivity of the anti-human ADAM2 antibodies, we carried out immunoprecipitation analyses using lysates from monkey testis. We were unable to obtain sufficient lysates from human testis due to limited sample availability. Anti-hADAM2-CyT effectively precipitated the ADAM2 protein from monkey testis lysates (Fig 8), whereas anti-hADAM2-Cys did not (data not shown), indicating that the latter antibody failed to recognize the native form of ADAM2. To investigate whether the ADAM2 is associated with other proteins in monkey testis, the anti-hADAM2-CyT immunoprecipitates were subjected to proteomic analysis by LC-MS/MS (n = 1) (Table 2). NRS was used as a negative control. Commonly detected proteins between anti-hADAM2-CyT and NRS were omitted in Table 2. Our results demonstrated that the precipitates contained ADAM2. Interestingly, this experiment further suggested that uncharacterized protein C19orf71 homolog, 78-kDa glucose-regulated protein (G8F3Q7), and heat shock 70-kDa protein 1-like (HS71L) could be potential ADAM2-interacting proteins (Table 2).


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)

Immunoprecipitation with anti-hADAM2-CyT.Immunoprecipitation of ADAM2 was carried out using monkey testis lysates. Immunoprecipitation using normal rabbit serum was performed as a negative control. Immunoprecipitated lysates were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gel. Abbreviations: TL, tissue lysate (100 μg); S, supernatant; and IP, immunoprecipitated protein (1 mg); and NRS, normal rabbit serum.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920383&req=5

pone.0158321.g008: Immunoprecipitation with anti-hADAM2-CyT.Immunoprecipitation of ADAM2 was carried out using monkey testis lysates. Immunoprecipitation using normal rabbit serum was performed as a negative control. Immunoprecipitated lysates were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gel. Abbreviations: TL, tissue lysate (100 μg); S, supernatant; and IP, immunoprecipitated protein (1 mg); and NRS, normal rabbit serum.
Mentions: In the course of analyzing the immunoreactivity of the anti-human ADAM2 antibodies, we carried out immunoprecipitation analyses using lysates from monkey testis. We were unable to obtain sufficient lysates from human testis due to limited sample availability. Anti-hADAM2-CyT effectively precipitated the ADAM2 protein from monkey testis lysates (Fig 8), whereas anti-hADAM2-Cys did not (data not shown), indicating that the latter antibody failed to recognize the native form of ADAM2. To investigate whether the ADAM2 is associated with other proteins in monkey testis, the anti-hADAM2-CyT immunoprecipitates were subjected to proteomic analysis by LC-MS/MS (n = 1) (Table 2). NRS was used as a negative control. Commonly detected proteins between anti-hADAM2-CyT and NRS were omitted in Table 2. Our results demonstrated that the precipitates contained ADAM2. Interestingly, this experiment further suggested that uncharacterized protein C19orf71 homolog, 78-kDa glucose-regulated protein (G8F3Q7), and heat shock 70-kDa protein 1-like (HS71L) could be potential ADAM2-interacting proteins (Table 2).

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