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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 rat testicular- and caudal sperm.100μg of testicular- or caudal- sperm protein was incubated for 2h without or with 300U of λ-PP at 30°C (A), or without or with 32U of CIP at 37°C (B) followed by NIA using 20ng of protein lysate thus treated to detect the GRP78 peaks. Figure shows representative isoelectropherograms for testicular- (Upper panel) and caudal- sperm (Lower panel). Figures in inset represent bar diagrams of the cumulative data. Values are mean ± Standard Deviation (SD). Statistical significance was determined using Paired Students’ ‘t’ test and significance level set at P ≤ 0.05.
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pone.0141858.g005: Phosphorylated forms of GRP78 in rat testicular- and caudal sperm.100μg of testicular- or caudal- sperm protein was incubated for 2h without or with 300U of λ-PP at 30°C (A), or without or with 32U of CIP at 37°C (B) followed by NIA using 20ng of protein lysate thus treated to detect the GRP78 peaks. Figure shows representative isoelectropherograms for testicular- (Upper panel) and caudal- sperm (Lower panel). Figures in inset represent bar diagrams of the cumulative data. Values are mean ± Standard Deviation (SD). Statistical significance was determined using Paired Students’ ‘t’ test and significance level set at P ≤ 0.05.

Mentions: To determine whether the different forms of GRP78 represented the different phosphorylation states of GRP78, Phosphatase assays were done. Incubation of testicular sperm with λ-PP demonstrated no change in any of the peaks (Fig 5A- Upper panel). The P values were0.19, 0.23 and 0.07 for GP4.94, GP4.96 and GP5.43, respectively. With caudal sperm, post λ-PP reaction, no change was observed in GP4.85 (P = 0.10), whereas GP4.94 showed complete reduction indicating this might be a phosphorylated form of GRP78 (P = 3.85 x 10−11). Significant increase was observed in GP4.96 (P = 3.5 x 10−11) and in GP5.43 (P = 5.4 x 10−5) (Fig 5A- Lower panel). Experiments were performed in 7–8 biological replicates with atleast 2 technical replicates for each. As complete dephosphorylation was not observed in caudal sperm and absolutely no dephosphorylation was observed in testicular sperm, we investigated whether the presence of phosphatase inhibitors used in the NP40 lysis buffer in any way influenced the GRP78 peak profiles of λ-PP treated testicular and caudal sperm lysates. The peak profile was similar regardless ofthe presence or absence of phosphatase inhibitors in the lysis buffer(S4 Fig). On CIP treatment of testicular sperm, peaks GP4.94 [P = 0.003] and GP4.96 [P = 0.0003] were significantly reduced indicating that these are indeed the phosphorylated forms of GRP78. GP5.43 [P = 0.0009] was significantly increased (Fig 5B-Upper panel). With caudal sperm, GP4.85 showed complete reduction [P = 7.02 x 10−6] and GP4.96 showed partial yet significant reduction [P = 0.02] indicating that these peak are phosphorylated forms of GRP78. Significant increase was observed in GP4.94 [P = 0.016] and in GP5.43 [P = 9.89 x 10−7], (Fig 5B–Lower panel). These assays were performed in two biological replicates with triplicates for each. Based on these observations, we believe that three phosphorylated forms of GRP78 are present in caudal sperm while in testicular sperm two phosphorylated forms exist. The observed pI for unphosphorylated GRP78 is 5.43.


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 rat testicular- and caudal sperm.100μg of testicular- or caudal- sperm protein was incubated for 2h without or with 300U of λ-PP at 30°C (A), or without or with 32U of CIP at 37°C (B) followed by NIA using 20ng of protein lysate thus treated to detect the GRP78 peaks. Figure shows representative isoelectropherograms for testicular- (Upper panel) and caudal- sperm (Lower panel). Figures in inset represent bar diagrams of the cumulative data. Values are mean ± Standard Deviation (SD). Statistical significance was determined using Paired Students’ ‘t’ test and significance level set at P ≤ 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664250&req=5

pone.0141858.g005: Phosphorylated forms of GRP78 in rat testicular- and caudal sperm.100μg of testicular- or caudal- sperm protein was incubated for 2h without or with 300U of λ-PP at 30°C (A), or without or with 32U of CIP at 37°C (B) followed by NIA using 20ng of protein lysate thus treated to detect the GRP78 peaks. Figure shows representative isoelectropherograms for testicular- (Upper panel) and caudal- sperm (Lower panel). Figures in inset represent bar diagrams of the cumulative data. Values are mean ± Standard Deviation (SD). Statistical significance was determined using Paired Students’ ‘t’ test and significance level set at P ≤ 0.05.
Mentions: To determine whether the different forms of GRP78 represented the different phosphorylation states of GRP78, Phosphatase assays were done. Incubation of testicular sperm with λ-PP demonstrated no change in any of the peaks (Fig 5A- Upper panel). The P values were0.19, 0.23 and 0.07 for GP4.94, GP4.96 and GP5.43, respectively. With caudal sperm, post λ-PP reaction, no change was observed in GP4.85 (P = 0.10), whereas GP4.94 showed complete reduction indicating this might be a phosphorylated form of GRP78 (P = 3.85 x 10−11). Significant increase was observed in GP4.96 (P = 3.5 x 10−11) and in GP5.43 (P = 5.4 x 10−5) (Fig 5A- Lower panel). Experiments were performed in 7–8 biological replicates with atleast 2 technical replicates for each. As complete dephosphorylation was not observed in caudal sperm and absolutely no dephosphorylation was observed in testicular sperm, we investigated whether the presence of phosphatase inhibitors used in the NP40 lysis buffer in any way influenced the GRP78 peak profiles of λ-PP treated testicular and caudal sperm lysates. The peak profile was similar regardless ofthe presence or absence of phosphatase inhibitors in the lysis buffer(S4 Fig). On CIP treatment of testicular sperm, peaks GP4.94 [P = 0.003] and GP4.96 [P = 0.0003] were significantly reduced indicating that these are indeed the phosphorylated forms of GRP78. GP5.43 [P = 0.0009] was significantly increased (Fig 5B-Upper panel). With caudal sperm, GP4.85 showed complete reduction [P = 7.02 x 10−6] and GP4.96 showed partial yet significant reduction [P = 0.02] indicating that these peak are phosphorylated forms of GRP78. Significant increase was observed in GP4.94 [P = 0.016] and in GP5.43 [P = 9.89 x 10−7], (Fig 5B–Lower panel). These assays were performed in two biological replicates with triplicates for each. Based on these observations, we believe that three phosphorylated forms of GRP78 are present in caudal sperm while in testicular sperm two phosphorylated forms exist. The observed pI for unphosphorylated GRP78 is 5.43.

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