kDa Phosphoprotein Research Articles

Evaluation of CMV DNA Antigenemia Status in Patients With Allogeneic Bone Marrow Transplant

Purpose: The risk of cytomegalovirus (CMV) reactivation following allogeneic hematopoietic stem cell transplantation (ASCT) reaches 30-50%, and there are numerous diagnostic tests to detect CMV replication. The most common tests used in this group of patients include 65kDa phosphoprotein (pp65) antigenemia immunofluorescence assay and nucleic-acid-based quantitative CMV-DNA polymerase chain reaction (qPCR). Material and Methods: In this study, patients who underwent ASCT and developed CMV positivity from 2009 to 2016 in our hospital were evaluated retrospectively. The study included samples of the same patient with antigenemia and CMV-DNA qPCR test for up to 48 hours. The study aimed to determine the factors affecting CMV DNA antigenemia and compare CMV DNA PCR and pp65 antigenemia immunofluorescence assay. Results: The results of 138 specimens of 39 patients who underwent ASCT were evaluated. The mean value of CMV PCR, which was positive for both tests, was 57.887 copies/ml (70- 1.213.633 copies/ml) and a significant correlation was found between the two tests and the positive samples (p = 0.018). The ROC analysis showed that 322 copies/ml CMV viral load in plasma corresponds to ≥1 antigen-positive cells/200 thousand leukocytes (Sensitivity: 68.5%; Specificity: 31.5%). CMV infection was observed in 32 samples; CMV DNA cut-off values of the reference according to CMV DNA PCR and antigenemia results, compared to the development of CMV infection, presented a significant correlation (p=0.004). Conclusion: Although there is a common agreement between antigenemia and CMV DNA PCR tests, one should keep in mind that the sensitivity of antigenemia test is low especially in the neutropenic period.

Trifluoperazine (TFP)-mediated fluorescence imaging approach reveals a probable calmodulin (CaM)-independent calcium signaling accompanying differential protein phosphorylation in NaCl-stressed sunflower seedlings (Helianthus annuus L. var. KBSH44)

The impact of calcium (Ca2+) under abiotic stress is manifested by both calmodulin (CaM)-dependent and calmodulin-independent pathways of signaling mechanisms in plants. Present work demonstrates the impact of NaCl (120 mM) stress on Ca2+-calmodulin activity and its spatial distribution in roots of 2d old sunflower seedlings (Helianthus annuus). NaCl stress significantly increases [Ca2+]cyt in roots (69%) which corresponds with 8% increase in activated calmodulin (Ca2+-CaM) levels. Thus, NaCl stress induced Ca2+ signaling in roots is likely to majorly operate through CaM-independent routes. Trifluoperazine-mediated fluorescence imaging reveals differential distribution of Ca2+-CaM complex in the elongation and differentiation zone of seedling roots which indicate spatial regulation of Ca2+-CaM signaling operative in the presence of NaCl stress. NaCl stress-induced CaM-independent Ca2+ signaling is further accompanied by differential protein phoshorylation of cytosolic proteins in seedling roots and cotyledons. Phosphoproteins exhibiting qualitative differences in roots in response to salt-stress were of both low and high molecular weight range (76–17.5 kDa) unlike in cotyledons where only high molecular weight (>76 kDa) phosphoproteins exhibited major differential expressions in response to 120 mM NaCl stress. Among the various polypeptides in the fraction of OB membrane proteins two polypeptides of 48 and >76 kDa exhibited increased protein phosphorylation in the presence of NaCl stress. NaCl stress for 48 h induces differences in the expression of phosphoproteins in roots and cotyledons, suggesting the long distance signaling of Na+ stress from roots to cotyledons. Roots seem to exhibit much stringent regulation of phosphoproteins in comparison with cotyledons which have lesser qualitative differences. Thus, present work provides evidence on the modulation of Ca2+-CaM signaling accompanying differential protein phosphorylation in sunflower seedling roots and cotyledons under NaCl stress.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

Cellular Immunity to Predict the Risk of Cytomegalovirus Infection in Kidney Transplantation: A Prospective, Interventional, Multicenter Clinical Trial.

Improving cytomegalovirus (CMV) immune-risk stratification in kidney transplantation is highly needed to establish guided preventive strategies. This prospective, interventional, multicenter clinical trial assessed the value of monitoring pretransplant CMV-specific cell-mediated immunity (CMI) using an interferon-γ release assay to predict CMV infection in kidney transplantation. One hundred sixty donor/recipient CMV-seropositive (D+/R+) patients, stratified by their baseline CMV (immediate-early protein 1)-specific CMI risk, were randomized to receive either preemptive or 3-month antiviral prophylaxis. Also, 15-day posttransplant CMI risk stratification and CMI specific to the 65 kDa phosphoprotein (pp65) CMV antigen were investigated. Immunosuppression consisted of basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids in 80% of patients, whereas 20% received thymoglobulin induction therapy. Patients at high risk for CMV based on pretransplant CMI developed significantly higher CMV infection rates than those deemed to be at low risk with both preemptive (73.3% vs 44.4%; odds ratio [OR], 3.44 [95% confidence interval {CI}, 1.30-9.08]) and prophylaxis (33.3% vs 4.1%; OR, 11.75 [95% CI, 2.31-59.71]) approaches. The predictive capacity for CMV-specific CMI was only found in basiliximab-treated patients for both preemptive and prophylaxis therapy. Fifteen-day CMI risk stratification better predicted CMV infection (81.3% vs 9.1%; OR, 43.33 [95% CI, 7.89-237.96]). Pretransplant CMV-specific CMI identifies D+/R+ kidney recipients at high risk of developing CMV infection if not receiving T-cell-depleting antibodies. Monitoring CMV-specific CMI soon after transplantation further defines the CMV infection prediction risk. Monitoring CMV-specific CMI may guide decision making regarding the type of CMV preventive strategy in kidney transplantation. NCT02550639.

A simple and efficient method of generating HCMV pp65-specific T cells using overlapping peptides.

The methods for expansion of human cytomegalovirus (HCMV)-specific T lymphocytes are limited due to the complex culture process, long culture duration, and human leukocyte antigen (HLA) restriction. Here, we report that in vitro stimulation with pp65kDa phosphoprotein (pp65)-derived overlapping synthetic peptides rapidly generates large numbers of HCMV-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) regardless of HLA type. Treatment of PBMCs from healthy volunteers expressing HLA-A*02:01 or HLA-A*24:02 with 138 pp65 overlapping peptides (OLP) resulted in an expansion of HCMV pp65 NLVPMVATV (NLV) pentamer-specific CD8+ T lymphocytes that expressed interferon (IFN)-γ, but the pp65 NLV peptide did not generate HCMV-specific CD8+ T lymphocytes in PBMCs obtained from an HLA-A*24:02 donor due to HLA restriction. The OLP-induced T lymphocytes specific for HCMV derived from PBMCs of HLA-A*02:01- and HLA-A*24:02-expressing donors showed effective cytolytic responses against target cells loaded with OLP or the NLV epitope, but pp65 NLV peptide-induced T lymphocytes did not. Phenotypic analyses demonstrated that OLP increased the frequency of CD3+ CD8+ cells, but not CD3+ CD4+, CD14+, or CD56+ cells, in donor PBMCs. Thus, this study provides evidence that in vitro stimulation with OLP efficiently generates sufficient numbers of HCMV pp65-specific cytotoxic T lymphocytes for adoptive cell therapy. Keywords: human cytomegalovirus; cytotoxic T lymphocyte; overlapping peptides; pp65; cytotoxicity.

Phosphoproteomics with Activated Ion Electron Transfer Dissociation.

The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript ( Anal. Chem. 2017 , DOI: 10.1021/acs.analchem.7b00213 ), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, an ∼23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.

The mitotic PP2A regulator ENSA/ARPP-19 is remarkably conserved across plants and most eukaryotes

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase of eukaryotes. PP2A containing the B55 subunit is a key regulator of mitosis and must be inhibited by phosphorylated α–endosulfine (ENSA) or cyclic AMP-regulated 19 kDa phosphoprotein (ARPP–19) to allow passage through mitosis. Exit from mitosis then requires dephosphorylation of ENSA/ARPP-19 to relieve inhibition of PP2A/B55. ENSA/ARPP-19 has been characterized in several vertebrates and budding yeast, but little is known about its presence in plants and the majority of other eukaryotes. Here we show that three isoforms of ENSA/ARPP-19 are present in the Arabidopsis thaliana genome with distinct expression profiles across various plant tissues. The ENSA/ARPP-19 proteins, and in particular their key inhibitory sequence FDSGDY (FDSADW in plants), is remarkably conserved across plants and most eukaryotes suggesting an ancient origin and conserved function to control PP2A activity.

Hormonal induction and roles of Disabled-2 in lactation and involution.

Disabled-2 (Dab2) is a widely expressed endocytic adaptor that was first isolated as a 96 KDa phospho-protein, p96, involved in MAPK signal transduction. Dab2 expression is lost in several cancer types including breast cancer, and Dab2 is thought to have a tumor suppressor function. In mammary epithelia, Dab2 was induced upon pregnancy and further elevated during lactation. We constructed mutant mice with a mosaic Dab2 gene deletion to bypass early embryonic lethality and to investigate the roles of Dab2 in mammary physiology. Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution. In primary cultures of mouse mammary epithelial cells, Dab2 proteins were also induced by estrogen, progesterone, and/or prolactin. Dab2 null mammary epithelial cells were refractory to growth suppression induced by TGF-beta. However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells. We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.

Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca(2+) as a second messenger. One of the proteins, the 46kDa phosphoprotein was found to be highly phosphorylated when Ca(2+)-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46kDa phosphoprotein was identified as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2',3'-cAMP (5μM) and, a non-competitive CNPase inhibitor, atractyloside (5μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca(2+)-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.

Regulation of α–endosulfine, an inhibitor of protein phosphatase 2A, by multisite phosphorylation

Progression into M phase requires inhibition of heterotrimeric PP2A containing the regulatory B55 subunit (PP2A-B55) as well as the activation of cyclin-dependent kinase 1 (Cdk1). α-endosulfine (ENSA)/cyclic AMP-regulated 19 kDa phosphoprotein (ARPP-19) family proteins phosphorylated at S67 by Greatwall kinase bind and inhibit PP2A-B55. This study shows that endogenous kinases phosphorylate not only S67 but also two additional sites in ENSA (T28 and S109) with different kinetics at different cell-cycle stages in Xenopus laevis intact cells and cell-free egg extracts. When assayed in vitro, these phosphorylations had qualitatively and/or quantitatively different effects on inhibition of PP2A-B55 by ENSA. Structural analyses revealed that the most-conserved middle region of ENSA containing S67 physically interacts with PP2A-B55 at the interface of the B55 and C subunits, where the catalytic centre of PP2A is located. As non-phosphorylated ENSA has an intrinsic potential for PP2A-B55 inhibition, these three phosphorylations differentially affect physical interaction of the middle region of ENSA with PP2A-B55. These results suggest that the two additional phosphorylation sites together with S67 allow ENSA to function as a 'stepwise tuner' for PP2A-B55, which may be regulated by multiple cellular signals, rather than a simple 'on/off' switch.

Exosomes derived from HTLV-1 infected cells contain the viral protein Tax

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator protein Tax has been identified as a critical component in the proliferation and transformation of human primary T-cells. This 40 kDa phosphoprotein not only manipulates chromatin remodeling within the host, but also subverts host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Here we utilized a combination of filtration and ultracentrifugation methods to enrich for exosomes from culture supernatants of HTLV-1 infected cells. We then employed western blots, mass spectrometry, and cytokine arrays to proteomically characterize the host and viral components in these exosomes. Additionally, RT-PCR was used to determine the presence of viral transcripts in these exosomes. Our results demonstrate that exosomes derived from HTLV-1 infected cells contain traditional exosome proteins. Furthermore, our proteomics studies revealed that these exosomes contain viral components such as gp46 and Tax, as well as inflammatory mediators including IL-6 and IL-10. We also investigated the presence of HTLV-1 mRNA transcripts of Env, Tax, HBZ, and 5’LTR contained within exosomes. Moreover, we evaluated the functional impacts of treating naive recipient cells with exosomes secreted from HTLV-1 infected cells, and determined that the exosomes were able to induce a response in reactive oxygen species production. Our observation that the viral protein Tax is contained within exosomes and may be transmitted in an extracellular capacity raises important implications to pathogenesis associated with HTLV-1 infections.

A novel 9 kDa phosphoprotein is a component of the primary cilium and interacts with polycystin-1

Polycystin-1 (PKD1) forms a mechanosensitive cation channel complex with polycystin-2 (PKD2) in the primary cilium. Functional defects in this complex caused by mutation of PKD1 and PKD2 result in autosomal dominant polycystic kidney disease (ADPKD). The mechanisms by which this complex regulates normal cell physiology remain elusive. In particular the proteins that interact directly with polycystin-1 remain poorly characterised. A Y-2H screen using the C-terminus of polycystin-1 as bait identified a novel binding partner that we have called PIP9 (polycystin interacting protein 9). PIP9 is evolutionary conserved, ubiquitously expressed, and predicted to be a 9KDa phosphoprotein containing a single coiled-coil domain that interacts with the coiled-coil domain of PC-1. Specific anti-PIP9 antisera confirmed the widespread and developmentally regulated expression of PIP9 and identified phosphorylated isoforms. PIP9 co-localised to the renal primary cilium with PC1 and immuno-EM studies revealed localisation to IFT particles. Knockdown of zPip9, using morpholino oligonucleotides, resulted in developmental defects in gastrulation movements. In addition, evidence of cell detachment was observed, common to the phenotype described for double bbs4;bbs10 morphant zebrafish which suggests pip9 may act in the same genetic pathway. zpip9 morphants also displayed variable head and eye degeneration, including coloboma and hydrocephalus, pronephric cysts and shortening of the body axis. Loss of Pip9 function in mice is lethal before embryonic day E11.5. This preliminary analysis suggests that PIP9 plays a central role in mediating polycystin-1 dependant signalling.

A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis

Reversible phosphorylation is a key signaling mechanism for modulating the functional properties of proteins involved in numerous cellular events. Abnormal protein phosphorylation causes many human diseases. Experimental procedures for the determination of the phosphorylation status of certain proteins are therefore very important in relation to studies on a diverse range of physiological and pathological processes. We have previously reported a novel phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique using a dizinc(II) complex of the phosphate-binding ligand Phos-tag in conjunction with a neutral-pH gel system to detect shifts in the mobilities of phosphoproteins (Zn2+–Phos-tag SDS-PAGE). However, this handmade gel-based procedure is often laborious and time-consuming to perform, and requires a skillful analyst. More recently, SuperSep Phos-tag precast gel has been developed on the basis of the Zn2+–Phos-tag SDS-PAGE methodology. This novel ready-to-use system employs a neutral-pH gel containing 12.5% (w/v) polyacrylamide and the immobilized Zn2+–Phos-tag (50 µM), which is generally used in conjunction with a Tris–glycine-based electrophoretic running buffer. We examined the potential usage of a Tris–N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tris–Tricine) buffer as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris–glycine, the Tris–Tricine running buffer improved the resolution of 8.8-35 kDa phosphoproteins and phosphopeptides. We can therefore provide a laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag affinity electrophoresis.

“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion

Abstract 682Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion. Disclosures:Leu:Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.

Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-(32)P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.

82 CRYOPRESERVATION OF BOAR EPIDIDYMAL SPERMATOZOA; ADDITION OF SEMINAL PLASMA TO THAWING SOLUTION IMPROVES REPRODUCTIVE PERFORMANCE BY ARTIFICIAL INSEMINATION

Epididymal spermatozoa are one of the available male germ cells for cryopreservation. It has been reported that frozen–thawed porcine epididymal spermatozoa have a high fertilization competence in vitro as compared with that in ejaculated one. However, there is little information about reproductive performance, such as conception rate or litter size, after artificial insemination (AI) using frozen–thawed epididymal spermatozoa. Recently, we demonstrated that the addition of seminal plasma to thawing solution improves membrane and acrosomal integrity, and enhanced both in vivo and in vitro fertilizing activity of frozen–thawed ejaculated spermatozoa. Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the number of implantation site (Okazaki et al. 2009 Theriogenology 71, 491–498). Thus, to apply those positive functions of seminal plasma to AI using frozen–thawed epididymal sperm, in this study, we added seminal plasma to thawing solution and then analysed the sperm functions including AI test using frozen–thawed epididymal spermatozoa. Epididymal spermatozoa collected by flushing caudal epididymis were frozen as described in our previous study (Okazaki et al. 2009). Frozen-spermatozoa were thawed in Modena solution with or without different percentages of seminal plasma. Protein tyrosine phosphorylation as a marker of capacitation was detected by western blotting. To examine the reproductive performance, the sows of natural oestrus were artificially inseminated two times (5 × 109 50 mL–1 per injection). When the frozen–thawed ejaculated or epididymal sperm was incubated up to 6 h, the motility of epididymal sperm was significantly higher than that of ejaculated sperm (19.6 v. 37.6%). However, the acrosomal membrane was damaged in epididymal sperm group at 3-h incubation period (15.2 v. 36.0%). The addition of seminal plasma [0, 10, 15, 20% (v/v)] in Modena solution protected the acrosomal injury (3 h; 35.2, 19.5, 15.6, 14.6%) and maintained high rate of motility (6 h; 38.8, 48.8, 62.5, 60.0%) in a dose-dependent manner. Furthermore, the addition of seminal plasma suppressed the expression of the 15 kDa phosphoprotein (early capacitation status), and the maximum effect was detected at 15% (v/v) seminal plasma. When the frozen–thawed epididymal spermatozoa with 15% (v/v) seminal plasma were artificially inseminated to swine (n = 15), the conception rate and the mean number of litter size were increased as compared with control (93 v. 43%, 10.0 v. 5.0). From these results, we concluded that the addition of seminal plasma to thawing solution was a beneficial method for artificial insemination using frozen–thawed epididymal spermatozoa in the pig. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Characterization of a low-molecular-mass stimulator protein of Mg2+-independent Ca2+-ATPase: effect on phosphorylation/dephosphorylation, calcium transport and sperm-cell motility

A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [gamma-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an alpha-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.

Letter: Correlation between Phosphorylation Ratios by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Analysis and Enzyme Kinetics

To identify the correlation between the phosphorylation ratios by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analysis and enzyme kinetics (Km, Vmax, and Vmax/Km) is important to understand whether MALDI-TOF MS can be applied for monitoring the properties of peptides that are substrates of protein kinases. The correlation between phosphorylation ratios and enzyme kinetics was examined using peptides for protein kinase C (PKC) and for 60 kDa phosphoprotein, encoded by the cellular sarcoma gene (c-Src). Phosphorylation ratios, analyzed by MALDI-ToF MS, showed higher correlation coefficient (r = or > +0.7) for Vmax/Km compared with that (r = or < -/+0.6) for Km or Vmax. For ion modes, a higher correlation coefficient between phosphorylation ratios and Vmax/Km was identified in the positive mode (r = or > +0.7) compared to that in the negative mode (r = or < +0.5). These results suggest that MALDI-ToF MS is a useful tool to evaluate Vmax/Km of peptides for protein kinases.

Visualization of shell matrix proteins in hemocytes and tissues of the Eastern oyster, Crassostrea virginica

The tissues of the oyster were examined for the presence of shell matrix proteins (SMPs) using a combination of Western, proteomic, and epi-fluorescent microscopy techniques. SMP, including 48 and 55 kDa phosphoproteins, was detected in the epithelial cells of mantle, gill, heart, and adductor muscle and linings of arteries and veins. The 48 kDa SMP circulates continuously within the hemolymph, and is present in the immune system hemocytes. It appears to be secreted from hemocytes on induction of shell repair. We suggest that the 48 and 55 kDa proteins are multifunctional and bridge the process of soft tissue repair and shell formation by mediating cellular activities during immune response as well as interacting with the mineral phase during deposition.