Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

<div><p>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, GP_4.96, GP_4.94 and GP_4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP_4.96and GP_4.94in immature (testicular) sperm. In mature human sperm GP_5.04, GP_4.96, and GP_4.94were the 3 phosphoforms observed. GP_4.94[P = 0.014]andGP_5.04 [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.</p></div

Multipleforms of GRP78 in Human sperm by NIA.

<p>Representative isoelectropherograms of NIA profile for GRP78 in normal sperm <b>(A)</b> and asthenozoosperm <b>(B)</b>. Four peaks GP_4.94, GP_4.96^, GP_5.04 and GP_5.43 are consistently observed. Bar diagram of the cumulative data comparing GRP78 profile of Asthenozoosperm with that of Normozoosperm is shown <b>(C)</b>. Experiments were performed in 3 biological replicates for each group with 3 technical replicates for each. Values expressed are mean ± Standard Deviation (SD). Statistical significance was determined using Unpaired Students’ ‘t’ test with significance level set at P ≤ 0.05. ‘GP’ represents the GRP78 Peak and the value in the subscript indicates the pI of the respective peak.</p

Phosphorylated forms of GRP78 in rat testicular- and caudal sperm.

<p>100μg of testicular- or caudal- sperm protein was incubated for 2h without or with 300U of λ-PP at 30°C (<b>A</b>), or without or with 32U of CIP at 37°C (<b>B</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.</p

Phosphorylated forms of GRP78 in human sperm.

<p>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 <b>(A)</b> and CIP <b>(B)</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 <b>(A)</b>. On CIP treatment (2h incubation), complete reduction was observed in GP_4.94 and significant reduction in GP_5.04. Significant increase was observed in GP_4.96 and GP_5.43<b>[Fig B; upper panel].</b>On overnight incubations with CIP, significant reduction was observed in GP_4.94 and GP_5.04. GP_4.96 was completely reduced and a significant increase was observed in the unphosphorylated form GP_5.43<b>[Fig B; lower panel].</b> 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.</p

GRP78 phosphorylation in rat testicular- and caudal sperm.

<p>(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>(B) Agarose bead Immunoprecipitation of Phosphoproteins.</b> 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.</p

GRP78 expression in sperm.

<p>A) Western blot analysis of GRP78 in human sperm, rat testis, and rat testicular- and caudal- sperm. GRP78 expression in human sperm (a) [Lane 1: negative control], and rat sperm and testis (b) [-ve: Negative control, C: Caudal sperm, T: Testicular sperm, Ts: Testicular tissue] B) IIF localization of GRP78. In normal human sperm, GRP78 expression seen in the equatorial region of sperm head, neck and midpiece when non-permeabilized (a) and on neck and midpiece when permeabilized (b). In caudal (mature) rat sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized (c) and on neck and anterior tip of sperm head on permeabilization (d) (Left panel). Inset shows the negative control for the respective images. DIC images of the respective sperm (Right panel)</p

SP-D and rhSP-D interfere with binding of gp120 to CD4.

<p>(<b>A</b>) Docked solution displaying SP-D timer docked with glycosylated model of HIV-1 glycoprotein gp120. (<b>i</b>) SP-D trimer (light green, dark green and orange cartoon) interacting with HIV-1 gp120 (grey cartoon) (<b>ii</b>) SP-D trimer (green) bound proximal to the CD4 binding region of gp120 (PDB ID: 1GC1). The CD4 (red cartoon) binding regions C2 (D loop), C4 and C5 of gp120 are depicted as light red, purple and cyan surfaces, respectively. Asn234 is depicted in blue and Asn276, a part of C2 domain is depicted in light red. (<b>iii</b>) Enlarged image of site where SP-D and CD4 interact on gp120 (<b>B</b>) To assess whether SP-D or rhSP-D interfered with gp120 and CD4 binding, PBMCs were incubated together with r-gp120 (2 µg/mL) and SP-D or rhSP-D (1 and 2 µg/ml) in 5 mM CaCl₂. The interaction was probed using a polyclonal FITC-tagged anti-gp120 antibody. Cells were acquired on a flow cytometer. (<b>i</b>) Lymphocyte population was gated. Differences in the FITC positive gated lymphocytes were calculated in terms of % inhibition. (<b>ii</b>) Mean fluorescence intensity was converted to % FITC positive cells. rhSP-D (at 1 µg 74.32±0.5%, at 2 µg 48.32±3.2%) showed a significant reduction in FITC positive cells as compared to untreated control. (iii) rhSP-D (at 10 µg–28.73±2.8%) showed a significant reduction in FITC positive cells as compared to untreated control. The data is a representative image of 4 independent experiments. A similar experiment was conducted using SP-D (data not shown) (<b>C</b>) Fusion experiment where HL2/3 cells that express gp120 were incubated with indicated concentrations rhSP-D or SP-D in presence of 5 mM CaCl₂. These cells were fused with TZM-bl cells that express CD4. The interaction of gp120 and CD4 is directly proportional to the blue color foci observed under a light microscope in TZM-bl cells. Each data point represents the mean ± S.D. (n = 3). Presence of SP-D and rhSP-D significantly decreased gp120 and CD4 binding and further β-gal positive TZM-bl cells.</p

rhSP-D inhibits pro-inflammatory cytokine production of HIV-1 challenged Jurkat T cells, U937 monocytic cells and activated PBMCs.

<p>Jurkat T cells, U937 monocytes and activated PBMCs were treated with indicated concentrations of rhSP-D, 20 min prior to HIV-1 challenge. 24 h post viral challenge, culture supernatant were collected and assayed for cytokine levels by a multiplex system. (<b>A</b>) Dose-dependent % inhibition of rhSP-D-treated HIV-1 challenged cytokines IL-2, TNF-α, IFN-γ, IL-1α, VEGF as compared to HIV alone in Jurkat T cells. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines. (<b>B</b>) Dose-dependent % inhibition of rhSP-D-treated HIV-1 challenged cytokines IL-2, IL-6, IFN-γ, MCP-1, VEGF as compared to HIV alone in U937 monocytes. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines. (<b>C</b>) Dose-dependent % inhibition of rhSP-D treated HIV challenged cytokines IL-2, IFN-γ, TNF-α, IL-1α, IL-1β, VEGF as compared to HIV alone. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines.</p

SP-D and rhSP-D recognize HIV-1 gp41, gp120 and gp160.

<p>(<b>A</b>) A representative image of a ligand blot analysis (n = 3) using a ready-to-use HIV-1 antigen PVDF strip, probed with SP-D or rhSP-D (1 µg/ml) in presence of 2 mM CaCl₂ and developed using a monoclonal antibody against human SP-D and a 1∶1000 anti-mouse IgG-peroxidase. SP-D and rhSP-D specifically bind to HIV-1 glycoproteins gp41, gp120 and gp 160. (<b>B</b>) ELISA assay showing the binding of different concentrations of SP-D (solid line) and rhSP-D (dotted line) to immobilized recombinant r-gp120 (2 µg/mL). SP-D or rhSP-D bind to gp120 in the presence of 5 mM CaCl₂, and the interaction is inhibited by 1 mM EDTA. Each data point represents the mean ± S.D. (n = 4). †, * shows a statistically significant increase in the binding of SP-D or rhSP-D respectively to HIV-1 gp120 in the presence of calcium compared to EDTA (p<0.05) (<b>C</b>) A representative histogram of FITC labeled gp120 on HL2/3 cells (n = 3). HIV-1 gp120 expressing HL2/3 cells were incubated with rhSP-D (2 µg/mL) or no rhSP-D and further probed with a polyclonal FITC-tagged anti-gp120 antibody. In presence of rhSP-D, significantly lesser FITC signal was detected by flow cytometry (gray tinted histogram) comparing to no rhSP-D (black lined histogram).</p

rhSP-D modulates the phosphorylation of Akt and MAPKs upon HIV-1 challenge.

<p>5×10⁵ Jurkat T cells or U937 monocytic cells were treated with rhSP-D (10 µg/ml) in presence of 5 mM CaCl₂ and HIV-1USNG31. After 30 min, cell were harvested, lysed, and SDS-PAGE and Western blots were performed using pAKT, p-p38 and p-Erk1/2 or β-actin (house-keeping control) antibodies. Representative Western blots (<b>A</b>) <b>and</b> (<b>C</b>) and bar graphs (<b>B</b>) <b>and</b> (<b>D</b>), generated by densitometric analysis for Jurkat and U937 cells, respectively. Each bar represents the mean ± S.D. (n = 3). *, ** shows statistical significant % inhibition cytokine levels of HIV-1 infection by rhSP-D in each cell type as compared to control (*p<0.05, **p<0.01) represent cumulative data.</p