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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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GRP78 phosphorylation in rat testicular- and caudal sperm.(A) GRP78 Immunoprecipitation. GRP78 immunoprecipitated from rat testicular- and caudal-sperm lysates was electrophoresed on 12% SDS-PAGE, transblotted and probed with Anti- GRP78 antibody(Lane 1–2) and Pan Phospho antibody (Lane 4–5). Lane 3: Input which was probed with Anti- GRP78 antibody. Rabbit IgG served as an isotype control for IP.(B) Agarose bead Immunoprecipitation of Phosphoproteins. Phosphoproteins were immunoprecipitated from 200μg of testicular-, or caudal sperm- lysates using either unconjugated agarose beads (control) or agarose beads conjugated anti Phospho-antibodies, electrophoresed on a 12% SDS-PAGE, transblotted and the blots were probed with Anti- GRP78 antibody. Lane 1: Control. Lanes 2–4: anti- Phosphoserine (anti- pS), anti- Phosphotyrosine (anti- pY), and anti- Phosphothreonine (anti- pT), respectively; Lane 5: Input.
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pone.0141858.g003: GRP78 phosphorylation in rat testicular- and caudal sperm.(A) GRP78 Immunoprecipitation. GRP78 immunoprecipitated from rat testicular- and caudal-sperm lysates was electrophoresed on 12% SDS-PAGE, transblotted and probed with Anti- GRP78 antibody(Lane 1–2) and Pan Phospho antibody (Lane 4–5). Lane 3: Input which was probed with Anti- GRP78 antibody. Rabbit IgG served as an isotype control for IP.(B) Agarose bead Immunoprecipitation of Phosphoproteins. Phosphoproteins were immunoprecipitated from 200μg of testicular-, or caudal sperm- lysates using either unconjugated agarose beads (control) or agarose beads conjugated anti Phospho-antibodies, electrophoresed on a 12% SDS-PAGE, transblotted and the blots were probed with Anti- GRP78 antibody. Lane 1: Control. Lanes 2–4: anti- Phosphoserine (anti- pS), anti- Phosphotyrosine (anti- pY), and anti- Phosphothreonine (anti- pT), respectively; Lane 5: Input.

Mentions: Immunoprecipitated GRP78 probed with a polyclonal Pan phospho antibody reveals that GRP78 is phosphorylated in rat testicular- and caudal- sperm (Fig 3A). Band seen in lane 2 represents GRP78 and that seen in lane 5 indicates its phosphorylation. A very weak band is also seen in lane 4. Phosphoproteins from rat sperm lysates immunoprecipitated using anti -Phosphoserine, -Phosphotyrosine and -Phosphothreonine antibodies when probed with GRP78 antibody shows GRP78 to be phosphorylated at serine, threonine and tyrosine residues in both testicular- as well as in caudal- sperm (Fig 3B). Proteins immunoprecipitated using unconjugated agarose beads when probed with GRP78 antibody does not show the GRP78 band.


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)

GRP78 phosphorylation in rat testicular- and caudal sperm.(A) GRP78 Immunoprecipitation. GRP78 immunoprecipitated from rat testicular- and caudal-sperm lysates was electrophoresed on 12% SDS-PAGE, transblotted and probed with Anti- GRP78 antibody(Lane 1–2) and Pan Phospho antibody (Lane 4–5). Lane 3: Input which was probed with Anti- GRP78 antibody. Rabbit IgG served as an isotype control for IP.(B) Agarose bead Immunoprecipitation of Phosphoproteins. Phosphoproteins were immunoprecipitated from 200μg of testicular-, or caudal sperm- lysates using either unconjugated agarose beads (control) or agarose beads conjugated anti Phospho-antibodies, electrophoresed on a 12% SDS-PAGE, transblotted and the blots were probed with Anti- GRP78 antibody. Lane 1: Control. Lanes 2–4: anti- Phosphoserine (anti- pS), anti- Phosphotyrosine (anti- pY), and anti- Phosphothreonine (anti- pT), respectively; Lane 5: Input.
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pone.0141858.g003: GRP78 phosphorylation in rat testicular- and caudal sperm.(A) GRP78 Immunoprecipitation. GRP78 immunoprecipitated from rat testicular- and caudal-sperm lysates was electrophoresed on 12% SDS-PAGE, transblotted and probed with Anti- GRP78 antibody(Lane 1–2) and Pan Phospho antibody (Lane 4–5). Lane 3: Input which was probed with Anti- GRP78 antibody. Rabbit IgG served as an isotype control for IP.(B) Agarose bead Immunoprecipitation of Phosphoproteins. Phosphoproteins were immunoprecipitated from 200μg of testicular-, or caudal sperm- lysates using either unconjugated agarose beads (control) or agarose beads conjugated anti Phospho-antibodies, electrophoresed on a 12% SDS-PAGE, transblotted and the blots were probed with Anti- GRP78 antibody. Lane 1: Control. Lanes 2–4: anti- Phosphoserine (anti- pS), anti- Phosphotyrosine (anti- pY), and anti- Phosphothreonine (anti- pT), respectively; Lane 5: Input.
Mentions: Immunoprecipitated GRP78 probed with a polyclonal Pan phospho antibody reveals that GRP78 is phosphorylated in rat testicular- and caudal- sperm (Fig 3A). Band seen in lane 2 represents GRP78 and that seen in lane 5 indicates its phosphorylation. A very weak band is also seen in lane 4. Phosphoproteins from rat sperm lysates immunoprecipitated using anti -Phosphoserine, -Phosphotyrosine and -Phosphothreonine antibodies when probed with GRP78 antibody shows GRP78 to be phosphorylated at serine, threonine and tyrosine residues in both testicular- as well as in caudal- sperm (Fig 3B). Proteins immunoprecipitated using unconjugated agarose beads when probed with GRP78 antibody does not show the GRP78 band.

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