The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP₃, GP₄ and GP₅ envelope glycoproteins, only the M/GP₅ glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP₅. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Proton Pump Inhibitors Exert Anti-Allergic Effects by Reducing TCTP Secretion

Background

Extracellular translationally controlled tumor protein (TCTP) is known to play a role in human allergic responses. TCTP has been identified outside of macrophages, in activated mononuclear cells, and in biological fluids from allergic patients. Even TCTP devoid of signal sequences, is secreted to extracellular environment by an yet undefined mechanism. This study is aimed at understanding the mechanism of TCTP release and its regulation. A secondary goal is to see if inhibitors of TCTP release can serve as potential anti-allergic asthmatic drugs.

Methodology/Principal Findings

Using Western blotting assay in HEK293 and U937 cells, we found that TCTP secretion is reduced by omeprazole and pantoprazole, both of which are proton pump inhibitors. We then transfected HEK293 cells with proton pump expression vectors to search for the effects of exogeneously overexpressed H⁺/K⁺-ATPase on the TCTP secretion. Based on these in vitro data we checked the in vivo effects of pantoprazole in a murine model of ovalbumin-induced allergy. Omeprazole and pantoprazole reduced TCTP secretion from HEK293 and U937 cells in a concentration-dependent fashion and the secretion of TCTP from HEK293 cells increased when they over-expressed H⁺/K⁺-ATPase. In a murine model of ovalbumin-induced allergy, pretreatment with pantoprazole reduced infiltration of inflammatory cells, increased goblet cells, and increased TCTP secretion induced by OVA challenge.

Conclusion

Since Omeprazole and pantoprazole decrease the secretion of TCTP which is associated with the development of allergic reaction, they may have the potential to serve as anti-allergic (asthmatic) drugs.     

Identification of the CD163 Protein Domains Involved in Infection of the Porcine Reproductive and Respiratory Syndrome Virus

Scavenger receptor CD163 is a key entry mediator for porcine reproductive and respiratory syndrome virus (PRRSV). To identify the CD163 protein domains involved in PRRSV infection, deletion mutants and chimeric mutants were created. Infection experiments revealed that scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR 5) is essential for PRRSV infection, while the four N-terminal SRCR domains and the cytoplasmic tail are not required. The remaining CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in reduced (SRCR 6 and interdomain regions) or unchanged (SRCR 7 to SRCR 9) infection efficiency. In addition, CD163-specific antibodies recognizing SRCR 5 are able to reduce PRRSV infection.Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating viral pig diseases worldwide (17, 26). The causative agent, PRRS virus (PRRSV), has a restricted host and cell tropism, with porcine alveolar macrophages as important target cells (7, 13, 25). PRRSV entry into these macrophages has been studied extensively (6, 15, 16, 28, 31), and to date, two macrophage-specific molecules are known as PRRSV entry mediators: the siglec sialoadhesin and scavenger receptor CD163 (2, 29, 30). The interaction between PRRSV and its internalization receptor, sialoadhesin, has been the subject of intensive investigation, with recently identification of the M/GP₅ complex as a viral ligand interacting with the N-terminal immunoglobulin-like domain of sialoadhesin (1, 4, 5, 27). In contrast, our understanding of the specific contribution of CD163 during PRRSV infection is still in its infancy. So far, it has been demonstrated that CD163 is not involved in virus binding and internalization in macrophages but most likely acts during PRRSV uncoating (30). Most recently, viral minor glycoproteins GP₂ and GP₄ were shown to interact with CD163 (3). Further, the two N-terminal scavenger receptor cysteine-rich (SRCR) domains are not involved, but the transmembrane domain is essential for CD163 to sustain PRRSV infection (2). To get more insight into the role of CD163 during PRRSV infection, this study aimed to identify the CD163 protein domains involved in PRRSV infection.     

ERp44 and ERGIC‐53 Synergize in Coupling Efficiency and Fidelity of IgM Polymerization and Secretion

Owing to the quality control mechanisms operating in the early secretory compartment, only native proteins are secreted. Despite the difficulties in assembling planar immunoglobulin M (IgM) polymers, antibody‐secreting cells can release up to thousands of IgM per second. The finding that secretory μ (μ_s) chains bind to ERGIC‐53, a lectin transporter that cycles in the early secretory compartment, suggested that ERGIC‐53 hexamers could provide a polymerization platform. Here, we show that ERGIC‐53 binds to the conserved Asn563 glycan in the C‐terminal μ_s tailpiece (μ_stp). Removal of this glycan inhibits ERGIC‐53 binding and results in the rapid formation of larger polymeric assemblies. In contrast, removal of the Asn402 oligosaccharides prevents both polymerization and secretion. ERp44, a chaperone that interacts with ERGIC‐53, binds to Cys575 in the μ_stp, providing a fail‐safe mechanism that retrieves unpolymerized IgM subunits and promotes polymerization. The coordinated action of ERGIC‐53 and ERp44 provides a way to improve the efficiency of IgM secretion without perturbing its fidelity.     

Thyroid hormone induces a 52 kDa soluble protein in goat testis Leydig cell which stimulates androgen release

Incubation of goat testicular Leydig cells with 3,5,3′-triiodothyronine(T₃) induces the generation of a proteinaceous factor (factors) which was located in the soluble supernatant fraction (100 000 × g supernatant, 100k sup) of sonicated Leydig cells. Addition of this factor to Leydig cell incubation greatly stimulated androgen release. This factor(s) was purified based on its biological properties, i.e., its addition to Leydig cell incubation augmented the release of androgen. This was designated as TIP (T₃-induced protein) activity. 100 k sup prepared from Leydig cells incubated in the absence (control) or presence of T₃ was gel filtrated through Sephadex G-100. 100 k sup from T₃ incubate gave two protein peaks, P-I and P-II, control 100 k sup had similar nature of P-I, but P-II was not well marked. Incubation in the presence of [¹⁴C]leucine clearly showed TCA precipitable radioactivity only in the P-II region of T₃ incubate. 5 μg of P-II protein stimulated androgen release from Leydig cells (1 · 10⁶ cells/well) to more than 5-fold as compared to control. P-II protein was further purifies by FPLC Mono-Q column chromatography where one unadsorbed (MQ-I) and two adsorbed(MQ-II and MQ-III) protein peaks could be detected. MQ-II, which was eluted with 0.20 M NaCl gradient, demonstrated strong TIP activity (2 μg protein released 4.8-fold more androgen as compared to control). MQ-II was passed through FPLC Superose-6 column where it gave two peaks and Peak-I(SP-I) showed strong TIP activity (2 μg protein stimulated a 6-fold increase in androgen release as compared to control). Polyacrylamide gel electrophoresis (PAGE) indicated SP-I to be a homogeneous protein and SDS-PAGE demonstrated it to be a 52 kDa monomer protein. Results show that T₃ induces the synthesis of a 52 kDa protein in testicular Leydig cells which in turn causes stimulation of androgen release suggesting this protein to be a novel mediator of T₃ function in Leydig cells.     

Pharmacological characterisation of the P2Y11 receptor in stably transfected haematological cell lines

In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn²⁺, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.     

Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion

Background

Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50–90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP_1,2) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP_1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP_1,2 trimeric, viral surface spike. After entry into host endosomes, GP_1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.

Principal Findings

We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP_1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP_1,2. Further, ELISA binding data of primed GP_1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.

Significance

The results reveal that the ebolavirus GP_1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP_1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.     

INFLUENCE OF SUCCESSIVE AND COMBINED ULTRAVIOLET A AND B IRRADIATIONS ON MATRIX METALLOELASTASES PRODUCED BY HUMAN DERMAL FIBROBLASTS IN CULTURE

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three-fold, at 24h intervals, with UVA (3×5J/cm²), UVB (3×8mJ/cm²), UVA plus UVB (3×5J/cm²and 3×8mJ/cm²) and the levels of 92kDa gelatinase (pro-MMP₉), 72kDa gelatinase (pro-MMP₂) and plasma-membrane elastase type protease were determined, following subsequent 24-h culture in 10% serum-containing medium. UV irradiations had only minor influence (1.4-fold increase for UVB) on secreted levels of pro-MMP₂and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66kDa activated MMP₂production: 2.5- and 1.7-fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro-MMP₉secretion by dermal fibroblast enhancing enzyme levels by 2.5-, 6.5- and 5-fold for UVA, UVB and UVA+UVB, respectively.     

Role of a putative tyrosine-O-sulfate receptor in the targeting and/or intracellular transport of tyrosine-sulfated proteins

By employing the affinity gel fraction technique, we have detected a 175 kDa tyrosine-O-sulfate (TyrS)-binding protein in sodium choleate extracts of the microsomal membrane fractions of bovine liver and pancreas, as well as canine liver and pancreas. Western blot analysis revealed the presence of the bovine liver TyrS-binding protein in complexes with tyrosine-sulfated proteins both in vivo and in vitro, suggesting the putative role of the former being the receptor for the latter. Using filter-grown Madin-Darby canine kidney (MDCK) cells as a model, it was demonstrated that the tyrosine-sulfated proteins synthesized were predominantly secreted into the apical medium. The results further indicate the production and differential polarized secretion of different sulfated forms of the two major secretory proteins produced by MDCK cells, fibronectin (FN) and an 80 kDa glycoprotein (gp 80), with their tyrosine-sulfated forms being predominantly secreted from the apical surface. Treatment of filter-grown MDCK cells with glycosylation inhibitors, swainsonine and 1-deoxymannojirimycin, appeared to enhance the apical secretion of tyrosine-sulfated FN and gp 80. A similar 175 kDa membrane-bound 'TyrS receptor', cross-reactive toward antiserum against the canine liver TyrS receptor, was shown to be present in MDCK cells. Pulse-chase experiments revealed its presence in complexes with newly synthesized FN and gp 80. A hypothetical model for TyrS residues serving as an apical targeting signal during the biosynthetic transport of tyrosine-sulfated proteins, as mediated by the TyrS receptor, in MDCK cells is proposed.     

SILAC‐based quantitative proteomic analysis of secretome of Marc‐145 cells infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV‐infected Marc‐145 cells compared with mock‐infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle‐mediated release of exosomal proteins, including different danger‐associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV‐infected Marc‐145 cells and provides valuable insight into the host response to PRRSV infection.     

The release of polypeptides and manganese from oxygen-evolving photosystem II preparations following zinc-treatment

Inhibition of oxygen evolution in photosystem II membrane fragments from pea chloroplasts by washing with Zn₂₊ causes appearance of the EPR signal of Mn(H₂O)₆²⁺. This Mn²⁺ remains associated with the membrane fraction. Release of Mn²⁺ into the medium was correlated with the amount of the 23 kDa protein removed from the membrane. This suggests that this protein may function as a ‘gate’ to an aqueous compartment into which Mn²⁺ is released. Inhibition by Zn²⁺ correlated with the release of 1 Mn²⁺ per reaction centre, out of a total stoichiometry of 4 Mn atoms per reaction centre. By comparing the release of Mn following Zn-treatment of NaCI or CaC1₂ washed membranes, it is concluded that the 33 kDa protein is involved in binding of 2 Mn.     

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12–O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt₂cAMP) augmented the secretion In contrast, TPA increased the level of type IIGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt₂cAMP affected neither IGF-l receptor number nor the IGFBP secretion. These results indicate that the production of IGF-l and the expression of IGF-l receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells. © 1994 Willey-Liss, Inc.     

A Comparative Analysis of the Molecular Features of MANF and CDNF

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔC_MANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔC_MANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNF_RTDL and CDNF_SARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNF_KTEL and CDNF_HPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANF_QTEL and MANF_YPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANF_KTEL and MANF_HPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F₁F_o-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc₁ complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F₁F_o-ATP synthase and NADH:ubiquinone oxidoreductase.     

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

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.     

Prosaposin sorting is mediated by oligomerization

The compartmental nature of eukaryotic cells requires sophisticated mechanisms of protein sorting. Prosaposin, the precursor of four sphingolipid activator proteins, is transported from the trans-Golgi network (TGN) to lysosomes as a partially glycosylated (65 kDa) protein with high-mannose/hybrid oligosaccharides. Prosaposin is also found in the extracellular space where it is secreted as a fully glycosylated (70 kDa) protein composed of complex glycans. Although the trafficking of prosaposin to lysosomes is known to be mediated by sortilin, the mechanism of secretion of this protein is still unknown. In this study, we report that prosaposin may covalently aggregate into oligomers. Our results demonstrate that while prosaposin oligomers are secreted into the extracellular space, monomeric prosaposin remains inside the cell bound to sortilin. We also found that deletion of the C-terminus of prosaposin, previously shown to block its lysosomal transport, did not abolish its oligomerization and secretion. On the other hand, elimination of the N-terminus and of each saposin domain inhibited its oligomerization and resulted in its retention as a fully glycosylated protein. In conclusion, we are reporting for the first time that oligomerization of prosaposin is crucial for its entry into the secretory pathway.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.