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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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Phosphorylated forms of GRP78 in human sperm.Phosphorylated forms of GRP78 in human sperm were discerned using λ-PP and CIP. Figure shows representative isoelectropherograms of NIA profiles for Normal human sperm post λ-PP (A) and CIP (B)treatment. 100μg of human sperm protein was incubated without or with 300U of λ-PP for 2h at 30°C. For CIP treatment, reactions were incubated for 2h or overnight at 37°C. 20ng of this protein was used for NIA. No significant change was observed in the peak profile post λ-PP reaction (A). On CIP treatment (2h incubation), complete reduction was observed in GP4.94 and significant reduction in GP5.04. Significant increase was observed in GP4.96 and GP5.43[Fig B; upper panel].On overnight incubations with CIP, significant reduction was observed in GP4.94 and GP5.04. GP4.96 was completely reduced and a significant increase was observed in the unphosphorylated form GP5.43[Fig B; lower panel]. Values are expressed as mean ± SD. Graphical representations of the cumulative data for GRP78 peak profiles in sperm post λ-PP / CIP treatments are shown.
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pone.0141858.g007: Phosphorylated forms of GRP78 in human sperm.Phosphorylated forms of GRP78 in human sperm were discerned using λ-PP and CIP. Figure shows representative isoelectropherograms of NIA profiles for Normal human sperm post λ-PP (A) and CIP (B)treatment. 100μg of human sperm protein was incubated without or with 300U of λ-PP for 2h at 30°C. For CIP treatment, reactions were incubated for 2h or overnight at 37°C. 20ng of this protein was used for NIA. No significant change was observed in the peak profile post λ-PP reaction (A). On CIP treatment (2h incubation), complete reduction was observed in GP4.94 and significant reduction in GP5.04. Significant increase was observed in GP4.96 and GP5.43[Fig B; upper panel].On overnight incubations with CIP, significant reduction was observed in GP4.94 and GP5.04. GP4.96 was completely reduced and a significant increase was observed in the unphosphorylated form GP5.43[Fig B; lower panel]. Values are expressed as mean ± SD. Graphical representations of the cumulative data for GRP78 peak profiles in sperm post λ-PP / CIP treatments are shown.

Mentions: The GRP78 profile for human sperm consistently showed 4 peaks GP4.94, GP4.96, GP5.04 and GP5.43 (Fig 6A and 6B). Analysis of the GRP78 profile of asthenozoosperm and normal sperm revealed that peaks GP4.94 [P = 0.014] and GP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. GP5.43 wassignificantly increased [P = 0.003] in asthenozoosperm compared to normal sperm. GP4.96 was comparable in asthenozoosperm and normal sperm [P = 0.93] (Fig 6C). λ-PP treatment of normal sperm shows no reduction in any of the peaks (Fig 7A). CIP treatment revealed that GP4.94, GP4.96 and GP5.04 are the phosphorylated forms of GRP78 (Fig 7B). Incubations with CIP for 2h showed complete reduction in GP4.94 [P = 0.0001] and significant decrease in GP5.04 [P = 0.0001]. Significant increase was observed in GP4.96 [P = 0.00005] and in GP5.43[P = 0.0006] (Fig 7B; Upper panel). On overnight incubations, significant reduction was observed in GP4.94 [P = 0.007] and GP5.04 [P = 0.005]. GP4.96 [P = 0.0003] showed complete reduction and significant increase was observed in GP5.43 [P = 0.0004] (B; Lower panel). Experiments were performed in 2 biological replicates with 3 technical replicates for each. Based on these phosphatase assays, we infer that GP4.94, GP4.96and GP5.04 are the three phosphorylated forms of GRP78 in human sperm. Reduction of peak intensities on CIP treatment suggests that in human sperm GRP78 may be predominantly tyrosine phosphorylated.


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)

Phosphorylated forms of GRP78 in human sperm.Phosphorylated forms of GRP78 in human sperm were discerned using λ-PP and CIP. Figure shows representative isoelectropherograms of NIA profiles for Normal human sperm post λ-PP (A) and CIP (B)treatment. 100μg of human sperm protein was incubated without or with 300U of λ-PP for 2h at 30°C. For CIP treatment, reactions were incubated for 2h or overnight at 37°C. 20ng of this protein was used for NIA. No significant change was observed in the peak profile post λ-PP reaction (A). On CIP treatment (2h incubation), complete reduction was observed in GP4.94 and significant reduction in GP5.04. Significant increase was observed in GP4.96 and GP5.43[Fig B; upper panel].On overnight incubations with CIP, significant reduction was observed in GP4.94 and GP5.04. GP4.96 was completely reduced and a significant increase was observed in the unphosphorylated form GP5.43[Fig B; lower panel]. Values are expressed as mean ± SD. Graphical representations of the cumulative data for GRP78 peak profiles in sperm post λ-PP / CIP treatments are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664250&req=5

pone.0141858.g007: Phosphorylated forms of GRP78 in human sperm.Phosphorylated forms of GRP78 in human sperm were discerned using λ-PP and CIP. Figure shows representative isoelectropherograms of NIA profiles for Normal human sperm post λ-PP (A) and CIP (B)treatment. 100μg of human sperm protein was incubated without or with 300U of λ-PP for 2h at 30°C. For CIP treatment, reactions were incubated for 2h or overnight at 37°C. 20ng of this protein was used for NIA. No significant change was observed in the peak profile post λ-PP reaction (A). On CIP treatment (2h incubation), complete reduction was observed in GP4.94 and significant reduction in GP5.04. Significant increase was observed in GP4.96 and GP5.43[Fig B; upper panel].On overnight incubations with CIP, significant reduction was observed in GP4.94 and GP5.04. GP4.96 was completely reduced and a significant increase was observed in the unphosphorylated form GP5.43[Fig B; lower panel]. Values are expressed as mean ± SD. Graphical representations of the cumulative data for GRP78 peak profiles in sperm post λ-PP / CIP treatments are shown.
Mentions: The GRP78 profile for human sperm consistently showed 4 peaks GP4.94, GP4.96, GP5.04 and GP5.43 (Fig 6A and 6B). Analysis of the GRP78 profile of asthenozoosperm and normal sperm revealed that peaks GP4.94 [P = 0.014] and GP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. GP5.43 wassignificantly increased [P = 0.003] in asthenozoosperm compared to normal sperm. GP4.96 was comparable in asthenozoosperm and normal sperm [P = 0.93] (Fig 6C). λ-PP treatment of normal sperm shows no reduction in any of the peaks (Fig 7A). CIP treatment revealed that GP4.94, GP4.96 and GP5.04 are the phosphorylated forms of GRP78 (Fig 7B). Incubations with CIP for 2h showed complete reduction in GP4.94 [P = 0.0001] and significant decrease in GP5.04 [P = 0.0001]. Significant increase was observed in GP4.96 [P = 0.00005] and in GP5.43[P = 0.0006] (Fig 7B; Upper panel). On overnight incubations, significant reduction was observed in GP4.94 [P = 0.007] and GP5.04 [P = 0.005]. GP4.96 [P = 0.0003] showed complete reduction and significant increase was observed in GP5.43 [P = 0.0004] (B; Lower panel). Experiments were performed in 2 biological replicates with 3 technical replicates for each. Based on these phosphatase assays, we infer that GP4.94, GP4.96and GP5.04 are the three phosphorylated forms of GRP78 in human sperm. Reduction of peak intensities on CIP treatment suggests that in human sperm GRP78 may be predominantly tyrosine phosphorylated.

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