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A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome.

Bhattacharya R, Devi MS, Dhople VM, Jesudasan RA - BMC Cell Biol. (2013)

Bottom Line: Novel proteins are still being reported from acrosome.MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome.The role of Y chromosome in the regulation of this complex is however not clear from the current study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. rachel@ccmb.res.in.

ABSTRACT

Background: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.

Results: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.

Conclusions: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

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Co-immunoprecipitation and Western blot analysis. (A) The upper panel shows the presence of caldendrin in IP complex of MAST antisera on probing with antisera raised to caldendrin. Signals of the same molecular weight are also present in the lysates (without immunoprecipitation) from mouse testis, kidney and brain indicating the presence of caldendrin in these tissues as well. In the lower panel IP with caldendrin antisera that is probed with MAST antisera shows the presence of MAST protein in the IP complex and testis lysate only. MAST is not present in other tissues such as kidney, spleen and brain. (B) Co-immunoprecipitation of MAST and calreticulin: Antisera of MAST used for IP and probed with calreticulin antibody shows the presence of calreticulin protein in the IP complex and the lysates from testis, kidney spleen and brain, with high expression in kidney. Calreticulin antibody used for immunoprecipitation and probed with antisera of MAST protein shows the presence of MAST protein in the IP complex and testis lysate only.
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Figure 4: Co-immunoprecipitation and Western blot analysis. (A) The upper panel shows the presence of caldendrin in IP complex of MAST antisera on probing with antisera raised to caldendrin. Signals of the same molecular weight are also present in the lysates (without immunoprecipitation) from mouse testis, kidney and brain indicating the presence of caldendrin in these tissues as well. In the lower panel IP with caldendrin antisera that is probed with MAST antisera shows the presence of MAST protein in the IP complex and testis lysate only. MAST is not present in other tissues such as kidney, spleen and brain. (B) Co-immunoprecipitation of MAST and calreticulin: Antisera of MAST used for IP and probed with calreticulin antibody shows the presence of calreticulin protein in the IP complex and the lysates from testis, kidney spleen and brain, with high expression in kidney. Calreticulin antibody used for immunoprecipitation and probed with antisera of MAST protein shows the presence of MAST protein in the IP complex and testis lysate only.

Mentions: Localization of MAST to the acrosome indicated possible functions in acrosome reaction/fertilization. In order to query the physiological role of MAST, interaction with other proteins localizing to the acrosome was studied by immunoprecipitation studies. Three acrosomal proteins considered in this study were caldendrin, acrosin and calreticulin. First, caldendrin was probed for interaction with MAST, if any. The immunopulldown (IP) using MAST antibody when probed with caldendrin antibody raised in the laboratory identified a signal on a western blot. When the pull down product of caldendrin antibody was probed with the MAST antibody also, a signal was identified that corresponded to the molecular weight of MAST from mouse testis and the overexpressed recombinant protein. Thus the Co-IP studies confirmed the interaction between caldendrin and MAST (Figure 4A). Co-IP studies using antibodies to calreticulin and MAST showed the presence of calreticulin in the pull down complex of MAST and vice versa; i.e. MAST antibody identified a signal on the western blot containing the complex pulled down with calreticulin antibody (Figure 4B). As caldendrin and calreticulin were both identified in the pulldown complex of MAST, this showed the presence of the three proteins in the same complex. Co-IP studies using MAST and acrosin antibodies showed that they did not interact (result not shown). These results show interaction of MAST with the calcium binding proteins caldendrin and calreticulin, but not with acrosin. Interaction of MAST was also tested with the testis-specific superoxide dismutase (SOD) that localizes on the mouse sperm tail as MAST. Co-IP using antibodies to MAST and SOD did not yield any signal (result not shown). This further confirms the interaction of MAST with the two calcium-binding proteins. The gene corresponding to caldendrin localizes to chromosome number 5 and that for calreticulin to chromosome number 8 in the mouse.


A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome.

Bhattacharya R, Devi MS, Dhople VM, Jesudasan RA - BMC Cell Biol. (2013)

Co-immunoprecipitation and Western blot analysis. (A) The upper panel shows the presence of caldendrin in IP complex of MAST antisera on probing with antisera raised to caldendrin. Signals of the same molecular weight are also present in the lysates (without immunoprecipitation) from mouse testis, kidney and brain indicating the presence of caldendrin in these tissues as well. In the lower panel IP with caldendrin antisera that is probed with MAST antisera shows the presence of MAST protein in the IP complex and testis lysate only. MAST is not present in other tissues such as kidney, spleen and brain. (B) Co-immunoprecipitation of MAST and calreticulin: Antisera of MAST used for IP and probed with calreticulin antibody shows the presence of calreticulin protein in the IP complex and the lysates from testis, kidney spleen and brain, with high expression in kidney. Calreticulin antibody used for immunoprecipitation and probed with antisera of MAST protein shows the presence of MAST protein in the IP complex and testis lysate only.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Co-immunoprecipitation and Western blot analysis. (A) The upper panel shows the presence of caldendrin in IP complex of MAST antisera on probing with antisera raised to caldendrin. Signals of the same molecular weight are also present in the lysates (without immunoprecipitation) from mouse testis, kidney and brain indicating the presence of caldendrin in these tissues as well. In the lower panel IP with caldendrin antisera that is probed with MAST antisera shows the presence of MAST protein in the IP complex and testis lysate only. MAST is not present in other tissues such as kidney, spleen and brain. (B) Co-immunoprecipitation of MAST and calreticulin: Antisera of MAST used for IP and probed with calreticulin antibody shows the presence of calreticulin protein in the IP complex and the lysates from testis, kidney spleen and brain, with high expression in kidney. Calreticulin antibody used for immunoprecipitation and probed with antisera of MAST protein shows the presence of MAST protein in the IP complex and testis lysate only.
Mentions: Localization of MAST to the acrosome indicated possible functions in acrosome reaction/fertilization. In order to query the physiological role of MAST, interaction with other proteins localizing to the acrosome was studied by immunoprecipitation studies. Three acrosomal proteins considered in this study were caldendrin, acrosin and calreticulin. First, caldendrin was probed for interaction with MAST, if any. The immunopulldown (IP) using MAST antibody when probed with caldendrin antibody raised in the laboratory identified a signal on a western blot. When the pull down product of caldendrin antibody was probed with the MAST antibody also, a signal was identified that corresponded to the molecular weight of MAST from mouse testis and the overexpressed recombinant protein. Thus the Co-IP studies confirmed the interaction between caldendrin and MAST (Figure 4A). Co-IP studies using antibodies to calreticulin and MAST showed the presence of calreticulin in the pull down complex of MAST and vice versa; i.e. MAST antibody identified a signal on the western blot containing the complex pulled down with calreticulin antibody (Figure 4B). As caldendrin and calreticulin were both identified in the pulldown complex of MAST, this showed the presence of the three proteins in the same complex. Co-IP studies using MAST and acrosin antibodies showed that they did not interact (result not shown). These results show interaction of MAST with the calcium binding proteins caldendrin and calreticulin, but not with acrosin. Interaction of MAST was also tested with the testis-specific superoxide dismutase (SOD) that localizes on the mouse sperm tail as MAST. Co-IP using antibodies to MAST and SOD did not yield any signal (result not shown). This further confirms the interaction of MAST with the two calcium-binding proteins. The gene corresponding to caldendrin localizes to chromosome number 5 and that for calreticulin to chromosome number 8 in the mouse.

Bottom Line: Novel proteins are still being reported from acrosome.MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome.The role of Y chromosome in the regulation of this complex is however not clear from the current study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. rachel@ccmb.res.in.

ABSTRACT

Background: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.

Results: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.

Conclusions: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

Show MeSH
Related in: MedlinePlus