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.
Highlights
Glucose Regulated Protein 78 (GRP78), a member of heat shock protein 70 family is a calcium sensitive chaperone induced upon calcium or glucose stress or energy deprivation and an anti-apoptotic protein [1, 2]
Using the Differential In Gel Electrophoresis (DIGE) approach, we demonstrate a shift in the electrophoretic mobility of phosphorylated GRP78 in asthenozoosperm vis-à-vis normozoosperm
Our findings suggest that 1) GRP78 phosphorylation in sperm undergoes a transformation during sperm maturation; 2) Two out of three GRP78 phosphoforms in human are significantly reduced in asthenozoosperm suggesting that these forms may have functional relevance in sperm motility
Summary
Glucose Regulated Protein 78 (GRP78), a member of heat shock protein 70 family is a calcium sensitive chaperone induced upon calcium or glucose stress or energy deprivation and an anti-apoptotic protein [1, 2]. Other than its chaperonic functions, GRP78 located on the cell surface is implicated in mitogenesis and cellular proliferation [3] and serves as a receptor for the uptake of certain viruses [4]. Surface expression of GRP78 has been reported on human sperm [6]. Gene and protein expression of GRP78 has been reported in pachytene and round spermatids during spermatogenesis [9]. Our studies investigating the phosphoproteins relevant to sperm motility demonstrated that whilst total GRP78 protein did not differ between normozoosperm and asthenozoosperm, it was significantly less phosphorylated in asthenozoosperm [12]. The present study was initiated to answer two questions; 1) Does sperm GRP78 undergo phosphorylation changes during epididymal maturation, 2) Are there any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm
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