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Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation.

Lobo V, Rao P, Gajbhiye R, Kulkarni V, Parte P - PLoS ONE (2015)

Bottom Line: Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa.GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm.Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Gamete Immunobiology, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400012, India.

ABSTRACT
GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

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Phosphorylation changes in GRP78 during sperm maturationImmature testicular sperm are known to undergo structural and functional maturation in the epididymis. 2 phosphorylated forms of GRP78 were observed in testicular (immature) sperm (GP4.94 and GP4.96) and 3 phosphorylated forms in caudal (mature) sperm (*GP4.85, GP4.94 and GP4.96). ‘*’ represents phosphorylated form of GRP78 exclusive to rat caudal sperm. Based on NIA profile and responses of rat testicular- and caudal—sperm GRP78 to phosphatases λ- PP and CIP, we hypothesize putative phosphorylation/ dephosphorylation changes in sperm GRP78 as the sperm traverses from the testis to caudal region of the epididymis and attains maturity. Testicular sperm represents immature sperm while the caudal sperm represents mature sperm. The remarkable changes are as follows;GP4.96 responds similarly to phosphatase treatment in both immature and mature sperm suggesting that GP4.96 is retained in the same form in the mature sperm as present in immature sperm (Black dashed arrows).GP4.96 increases in the mature caudal sperm. This may be due to a) phosphorylation of GP5.43 (Purple dashed arrow) which is substantiated by the observed decrease in GP5.43 in the mature sperm, or b) phosphorylation changes on GP4.94, in the immature sperm as it traverses through the epididymis (Blue dashed arrow).GP4.94 in mature sperm is probably not the same as in immature sperm as this form shows difference in response to phosphatase treatment in mature versus immature sperm indicating possible phosphorylation /dephosphorylation events happening in GP4.94 in immature sperm to give rise to new form of GP4.94 in mature sperm (Yellow dashed arrow).In the mature sperm, a) GP4.85 and GP4.96 are resistant to λ- PP but responsive to CIP, b) GP4.94 decreases significantly and a new GRP78 phosphoform GP4.85 emerges. Given that λ- PP is a serine / threonine phosphatase and CIP is a tyrosine specific phosphatase, we believe that GP4.94 in the immature sperm might undergo dephosphorylation on atleast 1 phosphorylated tyrosine(Y) residue, phosphorylation on atleast 1 serine/ threonine (S/ T) residue, and phosphorylation of predominantly tyrosine (Y) residues thus resulting in GP4.85 form in mature sperm (Orange dashed arrow).Graph on the left shows comparison of the respective peaks in testicular- (Tsp) and caudal- sperm (Cdsp). Number in parentheses indicates the number of amino acids phosphorylated.
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pone.0141858.g008: Phosphorylation changes in GRP78 during sperm maturationImmature testicular sperm are known to undergo structural and functional maturation in the epididymis. 2 phosphorylated forms of GRP78 were observed in testicular (immature) sperm (GP4.94 and GP4.96) and 3 phosphorylated forms in caudal (mature) sperm (*GP4.85, GP4.94 and GP4.96). ‘*’ represents phosphorylated form of GRP78 exclusive to rat caudal sperm. Based on NIA profile and responses of rat testicular- and caudal—sperm GRP78 to phosphatases λ- PP and CIP, we hypothesize putative phosphorylation/ dephosphorylation changes in sperm GRP78 as the sperm traverses from the testis to caudal region of the epididymis and attains maturity. Testicular sperm represents immature sperm while the caudal sperm represents mature sperm. The remarkable changes are as follows;GP4.96 responds similarly to phosphatase treatment in both immature and mature sperm suggesting that GP4.96 is retained in the same form in the mature sperm as present in immature sperm (Black dashed arrows).GP4.96 increases in the mature caudal sperm. This may be due to a) phosphorylation of GP5.43 (Purple dashed arrow) which is substantiated by the observed decrease in GP5.43 in the mature sperm, or b) phosphorylation changes on GP4.94, in the immature sperm as it traverses through the epididymis (Blue dashed arrow).GP4.94 in mature sperm is probably not the same as in immature sperm as this form shows difference in response to phosphatase treatment in mature versus immature sperm indicating possible phosphorylation /dephosphorylation events happening in GP4.94 in immature sperm to give rise to new form of GP4.94 in mature sperm (Yellow dashed arrow).In the mature sperm, a) GP4.85 and GP4.96 are resistant to λ- PP but responsive to CIP, b) GP4.94 decreases significantly and a new GRP78 phosphoform GP4.85 emerges. Given that λ- PP is a serine / threonine phosphatase and CIP is a tyrosine specific phosphatase, we believe that GP4.94 in the immature sperm might undergo dephosphorylation on atleast 1 phosphorylated tyrosine(Y) residue, phosphorylation on atleast 1 serine/ threonine (S/ T) residue, and phosphorylation of predominantly tyrosine (Y) residues thus resulting in GP4.85 form in mature sperm (Orange dashed arrow).Graph on the left shows comparison of the respective peaks in testicular- (Tsp) and caudal- sperm (Cdsp). Number in parentheses indicates the number of amino acids phosphorylated.

Mentions: On the basis of substrate specificities of the enzymes used, we can infer that in caudal sperm, GP4.94 may be serine / threonine phosphorylated while in testicular sperm it may not have a serine or threonine residue phosphorylated as treatment with λ-PP demonstrated complete reduction of GP4.94 for caudal sperm but not for testicular sperm suggesting that the phosphoform GP4.94 may not be the same in testicular- and caudal- sperm. However this same peak showed a significant reduction post CIP treatment. Phosphorylation changes in GRP78 during sperm maturation as decoded by us from our data are presented as a schematic illustration (Fig 8).


Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation.

Lobo V, Rao P, Gajbhiye R, Kulkarni V, Parte P - PLoS ONE (2015)

Phosphorylation changes in GRP78 during sperm maturationImmature testicular sperm are known to undergo structural and functional maturation in the epididymis. 2 phosphorylated forms of GRP78 were observed in testicular (immature) sperm (GP4.94 and GP4.96) and 3 phosphorylated forms in caudal (mature) sperm (*GP4.85, GP4.94 and GP4.96). ‘*’ represents phosphorylated form of GRP78 exclusive to rat caudal sperm. Based on NIA profile and responses of rat testicular- and caudal—sperm GRP78 to phosphatases λ- PP and CIP, we hypothesize putative phosphorylation/ dephosphorylation changes in sperm GRP78 as the sperm traverses from the testis to caudal region of the epididymis and attains maturity. Testicular sperm represents immature sperm while the caudal sperm represents mature sperm. The remarkable changes are as follows;GP4.96 responds similarly to phosphatase treatment in both immature and mature sperm suggesting that GP4.96 is retained in the same form in the mature sperm as present in immature sperm (Black dashed arrows).GP4.96 increases in the mature caudal sperm. This may be due to a) phosphorylation of GP5.43 (Purple dashed arrow) which is substantiated by the observed decrease in GP5.43 in the mature sperm, or b) phosphorylation changes on GP4.94, in the immature sperm as it traverses through the epididymis (Blue dashed arrow).GP4.94 in mature sperm is probably not the same as in immature sperm as this form shows difference in response to phosphatase treatment in mature versus immature sperm indicating possible phosphorylation /dephosphorylation events happening in GP4.94 in immature sperm to give rise to new form of GP4.94 in mature sperm (Yellow dashed arrow).In the mature sperm, a) GP4.85 and GP4.96 are resistant to λ- PP but responsive to CIP, b) GP4.94 decreases significantly and a new GRP78 phosphoform GP4.85 emerges. Given that λ- PP is a serine / threonine phosphatase and CIP is a tyrosine specific phosphatase, we believe that GP4.94 in the immature sperm might undergo dephosphorylation on atleast 1 phosphorylated tyrosine(Y) residue, phosphorylation on atleast 1 serine/ threonine (S/ T) residue, and phosphorylation of predominantly tyrosine (Y) residues thus resulting in GP4.85 form in mature sperm (Orange dashed arrow).Graph on the left shows comparison of the respective peaks in testicular- (Tsp) and caudal- sperm (Cdsp). Number in parentheses indicates the number of amino acids phosphorylated.
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Related In: Results  -  Collection

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Show All Figures
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pone.0141858.g008: Phosphorylation changes in GRP78 during sperm maturationImmature testicular sperm are known to undergo structural and functional maturation in the epididymis. 2 phosphorylated forms of GRP78 were observed in testicular (immature) sperm (GP4.94 and GP4.96) and 3 phosphorylated forms in caudal (mature) sperm (*GP4.85, GP4.94 and GP4.96). ‘*’ represents phosphorylated form of GRP78 exclusive to rat caudal sperm. Based on NIA profile and responses of rat testicular- and caudal—sperm GRP78 to phosphatases λ- PP and CIP, we hypothesize putative phosphorylation/ dephosphorylation changes in sperm GRP78 as the sperm traverses from the testis to caudal region of the epididymis and attains maturity. Testicular sperm represents immature sperm while the caudal sperm represents mature sperm. The remarkable changes are as follows;GP4.96 responds similarly to phosphatase treatment in both immature and mature sperm suggesting that GP4.96 is retained in the same form in the mature sperm as present in immature sperm (Black dashed arrows).GP4.96 increases in the mature caudal sperm. This may be due to a) phosphorylation of GP5.43 (Purple dashed arrow) which is substantiated by the observed decrease in GP5.43 in the mature sperm, or b) phosphorylation changes on GP4.94, in the immature sperm as it traverses through the epididymis (Blue dashed arrow).GP4.94 in mature sperm is probably not the same as in immature sperm as this form shows difference in response to phosphatase treatment in mature versus immature sperm indicating possible phosphorylation /dephosphorylation events happening in GP4.94 in immature sperm to give rise to new form of GP4.94 in mature sperm (Yellow dashed arrow).In the mature sperm, a) GP4.85 and GP4.96 are resistant to λ- PP but responsive to CIP, b) GP4.94 decreases significantly and a new GRP78 phosphoform GP4.85 emerges. Given that λ- PP is a serine / threonine phosphatase and CIP is a tyrosine specific phosphatase, we believe that GP4.94 in the immature sperm might undergo dephosphorylation on atleast 1 phosphorylated tyrosine(Y) residue, phosphorylation on atleast 1 serine/ threonine (S/ T) residue, and phosphorylation of predominantly tyrosine (Y) residues thus resulting in GP4.85 form in mature sperm (Orange dashed arrow).Graph on the left shows comparison of the respective peaks in testicular- (Tsp) and caudal- sperm (Cdsp). Number in parentheses indicates the number of amino acids phosphorylated.
Mentions: On the basis of substrate specificities of the enzymes used, we can infer that in caudal sperm, GP4.94 may be serine / threonine phosphorylated while in testicular sperm it may not have a serine or threonine residue phosphorylated as treatment with λ-PP demonstrated complete reduction of GP4.94 for caudal sperm but not for testicular sperm suggesting that the phosphoform GP4.94 may not be the same in testicular- and caudal- sperm. However this same peak showed a significant reduction post CIP treatment. Phosphorylation changes in GRP78 during sperm maturation as decoded by us from our data are presented as a schematic illustration (Fig 8).

Bottom Line: Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa.GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm.Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Gamete Immunobiology, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400012, India.

ABSTRACT
GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

Show MeSH
Related in: MedlinePlus