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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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Related in: MedlinePlus

Differential expression of phosphorylated GRP78 in asthenozoosperm.A) Phosphoprotein enrichment. 10μg of phosphorylated (P10) and unphosphorylated (UP10) fractions of sperm lysates were electrophoresed on 1D SDS-PAGE and stained with Pro-Q Diamond Phosphoprotein gel stain which stains only phosphoproteins (a) and subsequently with SYPRO® Ruby which stains all proteins (b). This phosphoprotein enrichment was done for both normal and asthenozoospermic samples. PM: Peppermint phospho marker, RPN756: was the protein marker used (GE Healthcare Life Sciences, UK). B) A representative DIGE gel showing the profile of a few proteins in normal sperm and asthenozoosperm. The image on the left represents a snapshot of a fewphosphoproteins from normal sperm (Cy 3 labelled) and one on the right represents the same snapshot but that of asthenozoosperm (Cy 5 labelled). The arrow indicates identity of the spot as GRP78. Dotted arrow in the snapshot for asthenozoosperm points to the shifted location of phosphorylated GRP78 to the basic side suggesting less phosphorylation as compared to normal.
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pone.0141858.g001: Differential expression of phosphorylated GRP78 in asthenozoosperm.A) Phosphoprotein enrichment. 10μg of phosphorylated (P10) and unphosphorylated (UP10) fractions of sperm lysates were electrophoresed on 1D SDS-PAGE and stained with Pro-Q Diamond Phosphoprotein gel stain which stains only phosphoproteins (a) and subsequently with SYPRO® Ruby which stains all proteins (b). This phosphoprotein enrichment was done for both normal and asthenozoospermic samples. PM: Peppermint phospho marker, RPN756: was the protein marker used (GE Healthcare Life Sciences, UK). B) A representative DIGE gel showing the profile of a few proteins in normal sperm and asthenozoosperm. The image on the left represents a snapshot of a fewphosphoproteins from normal sperm (Cy 3 labelled) and one on the right represents the same snapshot but that of asthenozoosperm (Cy 5 labelled). The arrow indicates identity of the spot as GRP78. Dotted arrow in the snapshot for asthenozoosperm points to the shifted location of phosphorylated GRP78 to the basic side suggesting less phosphorylation as compared to normal.

Mentions: DIGE was performed following the protocol described by Yan’s group [15]. Sperm from normal- and asthenozoospermic individuals were processed for Differential In Gel Electrophoresis (DIGE) after phosphoprotein enrichment performed exactly as described earlier [12]. The protein content was quantified using the modified BCA assay. Enrichment of the phosphorylated proteins was confirmed by electrophoresing10μg of phosphorylated and unphosphorylated fractions on 1D SDS-PAGE gels and staining the gel with Pro-Q Diamond Phosphoprotein gel stain (Invitrogen, CA, USA) and subsequently with SYPRO® Ruby (Molecular probes, Eugene, OR) (Fig 1A). 50μg phosphoprotein from each normal and asthenozoospermic individual were labeled with 100pmol of Cy3 and Cy5, respectively. Internal standard was prepared by pooling equal amount of protein from each of the samples in the experiment and 50μg of this was labeled with Cy2 dye, as per the kit protocol (Cy dye minimal labeling kit, GE Healthcare). The Cy 3 -, Cy 5 -, and Cy 2—labeled samples were then pooled and passively rehydrated on a 13 cm 3–10 NL IPG strip and focused using a EttanIPGPhor II focusing unit (GE Healthcare) to resolve the proteins on the basis of their isoelectric point (pI). Towards this, active rehydration was done at 50V for 10h, pre-focusing at 200V, 500V, 1000V, and 2500V for 1h each, followed by isoelectric focusing at a gradient 6000V for 20000Vh, and 7000V for 0.5h. Following focusing, the IPG strip was equilibrated in 0.5% w/v DTT and 2.5% w/v iodoacetamide, sequentially in equilibration buffer containing 6M urea, 2% SDS, 50 mM Tris, pH 8.4, and 30% glycerol. The proteins on the strips were further resolved on the basis of their molecular size on 12% SDS-PAGE. The images were acquired on Ettan DIGE imager at excitation wavelengths of 532nm for Cy3, 633nm for Cy5 and 488nm for Cy2. All gels were scanned at a resolution of 100μm. Analysis of the differentially expressed protein spots was done using the DeCyder 6.5 software (GE Healthcare). As the phosphorylated fractions were too little to run preparatory gels, protein spots of interest were picked robotically (InvestigatorTMProPic II, Genomic Solutions, UK) from the analytical gels, processed for trypsin digestion, and analyzed by MALDI-TOF-MS/MS in the reflectron mode using a 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Illinois, US). The protocol followed was as described earlier [16]. Briefly, protein spots of interest (as identified from DIGE analysis using DeCyder Software) were picked robotically from the analytical gels and digested in-gel with trypsin protease and extracted with trifluoroacetic acid (TFA) and acetonitrile (ACN). The spots were alternately treated with 25mM ammonium bicarbonate and 50% ACN, twice followed by a final treatment with 100% ACN and incubated with 150ng of trypsin overnight at 37°C. The in-gel digested peptides were eluted with grades of 0.1%, 0.5%, and 1% TFA in 50% ACN. Approximately 0.5μl tryptic peptide was mixed with 0.5μl α-cyano-4-hydroxycinnamic acid matrix, and 0.5μl of this mixture was spotted onto the target plate. MALDI-TOF-MS/MS was performed in the reflectron mode. Combined MS and MS/MS spectra were subjected to protein identification against the taxonomy Mammalia (mammals) in the Swiss-Prot Protein knowledgebase (Swiss-Prot Release 20080226; 356194 sequences; 127836513 residues) and / NCBInrDataBase (Release 20070216; 4626804 sequences; 1596079197 residues) using the GPS software (version 3.5, Applied Biosystems) running Mascot search algorithm (version 2.0, Matrix Science, Boston, MA) for peptide and protein identification. For peptide matching, a maximum of one miscleavage per peptide and peptide modifications by oxidation of Met and carbamidomethylation of Cys were allowed. The peptide mass tolerance and ion mass (MS/MS) accuracy used for peptide matching were 100 ppm and 0.25 Da, respectively. The confidence of peptide matches was based on the significant value of the MOWSE score and the percentage of sequence coverage.


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)

Differential expression of phosphorylated GRP78 in asthenozoosperm.A) Phosphoprotein enrichment. 10μg of phosphorylated (P10) and unphosphorylated (UP10) fractions of sperm lysates were electrophoresed on 1D SDS-PAGE and stained with Pro-Q Diamond Phosphoprotein gel stain which stains only phosphoproteins (a) and subsequently with SYPRO® Ruby which stains all proteins (b). This phosphoprotein enrichment was done for both normal and asthenozoospermic samples. PM: Peppermint phospho marker, RPN756: was the protein marker used (GE Healthcare Life Sciences, UK). B) A representative DIGE gel showing the profile of a few proteins in normal sperm and asthenozoosperm. The image on the left represents a snapshot of a fewphosphoproteins from normal sperm (Cy 3 labelled) and one on the right represents the same snapshot but that of asthenozoosperm (Cy 5 labelled). The arrow indicates identity of the spot as GRP78. Dotted arrow in the snapshot for asthenozoosperm points to the shifted location of phosphorylated GRP78 to the basic side suggesting less phosphorylation as compared to normal.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141858.g001: Differential expression of phosphorylated GRP78 in asthenozoosperm.A) Phosphoprotein enrichment. 10μg of phosphorylated (P10) and unphosphorylated (UP10) fractions of sperm lysates were electrophoresed on 1D SDS-PAGE and stained with Pro-Q Diamond Phosphoprotein gel stain which stains only phosphoproteins (a) and subsequently with SYPRO® Ruby which stains all proteins (b). This phosphoprotein enrichment was done for both normal and asthenozoospermic samples. PM: Peppermint phospho marker, RPN756: was the protein marker used (GE Healthcare Life Sciences, UK). B) A representative DIGE gel showing the profile of a few proteins in normal sperm and asthenozoosperm. The image on the left represents a snapshot of a fewphosphoproteins from normal sperm (Cy 3 labelled) and one on the right represents the same snapshot but that of asthenozoosperm (Cy 5 labelled). The arrow indicates identity of the spot as GRP78. Dotted arrow in the snapshot for asthenozoosperm points to the shifted location of phosphorylated GRP78 to the basic side suggesting less phosphorylation as compared to normal.
Mentions: DIGE was performed following the protocol described by Yan’s group [15]. Sperm from normal- and asthenozoospermic individuals were processed for Differential In Gel Electrophoresis (DIGE) after phosphoprotein enrichment performed exactly as described earlier [12]. The protein content was quantified using the modified BCA assay. Enrichment of the phosphorylated proteins was confirmed by electrophoresing10μg of phosphorylated and unphosphorylated fractions on 1D SDS-PAGE gels and staining the gel with Pro-Q Diamond Phosphoprotein gel stain (Invitrogen, CA, USA) and subsequently with SYPRO® Ruby (Molecular probes, Eugene, OR) (Fig 1A). 50μg phosphoprotein from each normal and asthenozoospermic individual were labeled with 100pmol of Cy3 and Cy5, respectively. Internal standard was prepared by pooling equal amount of protein from each of the samples in the experiment and 50μg of this was labeled with Cy2 dye, as per the kit protocol (Cy dye minimal labeling kit, GE Healthcare). The Cy 3 -, Cy 5 -, and Cy 2—labeled samples were then pooled and passively rehydrated on a 13 cm 3–10 NL IPG strip and focused using a EttanIPGPhor II focusing unit (GE Healthcare) to resolve the proteins on the basis of their isoelectric point (pI). Towards this, active rehydration was done at 50V for 10h, pre-focusing at 200V, 500V, 1000V, and 2500V for 1h each, followed by isoelectric focusing at a gradient 6000V for 20000Vh, and 7000V for 0.5h. Following focusing, the IPG strip was equilibrated in 0.5% w/v DTT and 2.5% w/v iodoacetamide, sequentially in equilibration buffer containing 6M urea, 2% SDS, 50 mM Tris, pH 8.4, and 30% glycerol. The proteins on the strips were further resolved on the basis of their molecular size on 12% SDS-PAGE. The images were acquired on Ettan DIGE imager at excitation wavelengths of 532nm for Cy3, 633nm for Cy5 and 488nm for Cy2. All gels were scanned at a resolution of 100μm. Analysis of the differentially expressed protein spots was done using the DeCyder 6.5 software (GE Healthcare). As the phosphorylated fractions were too little to run preparatory gels, protein spots of interest were picked robotically (InvestigatorTMProPic II, Genomic Solutions, UK) from the analytical gels, processed for trypsin digestion, and analyzed by MALDI-TOF-MS/MS in the reflectron mode using a 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Illinois, US). The protocol followed was as described earlier [16]. Briefly, protein spots of interest (as identified from DIGE analysis using DeCyder Software) were picked robotically from the analytical gels and digested in-gel with trypsin protease and extracted with trifluoroacetic acid (TFA) and acetonitrile (ACN). The spots were alternately treated with 25mM ammonium bicarbonate and 50% ACN, twice followed by a final treatment with 100% ACN and incubated with 150ng of trypsin overnight at 37°C. The in-gel digested peptides were eluted with grades of 0.1%, 0.5%, and 1% TFA in 50% ACN. Approximately 0.5μl tryptic peptide was mixed with 0.5μl α-cyano-4-hydroxycinnamic acid matrix, and 0.5μl of this mixture was spotted onto the target plate. MALDI-TOF-MS/MS was performed in the reflectron mode. Combined MS and MS/MS spectra were subjected to protein identification against the taxonomy Mammalia (mammals) in the Swiss-Prot Protein knowledgebase (Swiss-Prot Release 20080226; 356194 sequences; 127836513 residues) and / NCBInrDataBase (Release 20070216; 4626804 sequences; 1596079197 residues) using the GPS software (version 3.5, Applied Biosystems) running Mascot search algorithm (version 2.0, Matrix Science, Boston, MA) for peptide and protein identification. For peptide matching, a maximum of one miscleavage per peptide and peptide modifications by oxidation of Met and carbamidomethylation of Cys were allowed. The peptide mass tolerance and ion mass (MS/MS) accuracy used for peptide matching were 100 ppm and 0.25 Da, respectively. The confidence of peptide matches was based on the significant value of the MOWSE score and the percentage of sequence coverage.

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