A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.     

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.     

Bovine cells expressing bovine herpesvirus 1 (BHV-1) glycoprotein IV resist infection by BHV-1, herpes simplex virus, and pseudorabies virus.

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.     

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus.

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.     

Bovine herpesvirus type 1 (BHV-1) UL49.5 luminal domain residues 30 to 32 are critical for MHC-I down-regulation in virus-infected cells

Bovine herpesvirus type 1 (BHV-1) U(L)49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. We have constructed a BHV-1 U(L)49.5 cytoplasmic tail (CT) null and several U(L)49.5 luminal domain mutants in the backbone of wild-type BHV-1 or BHV-1 U(L)49.5 CT- null viruses and determined their relative TAP mediated peptide transport inhibition and MHC-1 down-regulation properties compared with BHV-1 wt. Based on our results, the U(L)49.5 luminal domain residues 30-32 and U(L)49.5 CT residues, together, promote efficient TAP inhibition and MHC-I down-regulation functions. In vitro, BHV-1 U(L)49.5 Δ30-32 CT-null virus growth property was similar to that of BHV-1 wt and like the wt U(L)49.5, the mutant U(L)49.5 was incorporated in the virion envelope and it formed a complex with gM in the infected cells.     

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin-Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-x(L) decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol.     

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).     

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby Bovine Kidney (MDBK) cells in vitro

Dioxin-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type-1 (BHV-1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV-1 infection, in Madin-Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV-1 showed a dose-dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV-1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV-1 titer, TCDD may contribute to reactivate BHV-1 from latency, leading to recurrent disease and increase virus transmission.     

CASP9 (caspase 9) is essential for autophagosome maturation through regulation of mitochondrial homeostasis

ABSTRACT

CASP9 (caspase 9) is a well-known initiator caspase which triggers intrinsic apoptosis. Recent studies also suggest various non-apoptotic roles of CASP9, including macroautophagy/autophagy regulation. However, the involvement of CASP9 in autophagy and its molecular mechanisms are not well understood. Here we report the non-apoptotic function of CASP9 in positive regulation of autophagy through maintenance of mitochondrial homeostasis. Growth factor or amino acid deprivation-induced autophagy activated CASP9, but without apoptotic features. Pharmacological inhibition or genetic ablation of CASP9 decreased autophagy flux, while ectopic expression of CASP9 rescued autophagy defects. In CASP9 knockout (KO) cells, initiation and elongation of phagophore membranes were normal, but sealing of the membranes and autophagosome maturation were impaired, and the lifetime of autophagosomes was prolonged. Ablation of CASP9 caused an accumulation of inactive ATG3 and decreased lipidation of the Atg8-family members, most severely that of GABARAPL1. Moreover, it resulted in abnormal mitochondrial morphology with depolarization of the membrane potential, reduced reactive oxygen species production, and aberrant accumulation of mitochondrial fusion-fission proteins. CASP9 expression or exogenously added H₂O₂ in the CASP9 KO cells corrected the ATG3 level and lipidation status of Atg8-family members, and restored autophagy flux. Of note, only CASP9 expression but not H₂O₂ rescued mitochondrial defects, revealing regulation of mitochondrial homeostasis by CASP9. Our findings suggest a new regulatory link between mitochondria and autophagy through CASP9 activity, especially for the proper operation of the Atg8-family conjugation system and autophagosome closure and maturation.     

Bovine herpesvirus 1 U(L)3.5 interacts with bovine herpesvirus 1 alpha-transinducing factor

The bovine herpesvirus 1 (BHV-1) U(L)3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U(L)3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U(L)3.5 is deficient in viral egress but can be complemented by BHV-1 U(L)3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). The function of BHV-1 U(L)3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U(L)3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 alpha-transinducing factor (alphaBTIF) as a BHV-1 U(L)3. 5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U(L)3.5 and alphaBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U(L)3.5, but not 40 amino acids from the C terminus, abolished the U(L)3.5-alphaBTIF interaction both in vitro and in vivo. The interaction between U(L)3. 5 and alphaBTIF may be important for BHV-1 maturation and regulation of alphaBTIF transactivation activity.     

Transactivator protein BICP0 of bovine herpesvirus 1 (BHV-1) is blocked by prostaglandin D2 (PGD2), which points to a mechanism for PGD2-mediated inhibition of BHV-1 replication

The immediate-early protein, BICP0, of bovine herpesvirus 1 (BHV-1) transactivates a variety of viral and cellular genes. In a yeast two-hybrid cDNA library screening, we found that lipocalin-type prostaglandin D synthase, which catalyzes the production of prostaglandin D(2) (PGD(2)), is a cellular target of BICP0. We observed that, during wild-type BHV-1 infection, PGD(2) levels were increased intracellularly and decreased in the medium. These effects were absent upon infection with recombinant BHV-1 expressing beta-galactosidase instead of BICP0 (A2G2). Transient-expression assays showed that BICP0 alone caused a significant increase in PGD(2) levels in the cell. PGD(2) repressed BHV-1 replication in cultured cells. Antiviral activities of prostaglandins have been documented long ago, but their mode of action remains to be clarified. Here we provide evidence that PGD(2) impairs the transactivation ability of BICP0 that is necessary for efficient virus replication.     

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.     

Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.     

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.     

A comparison of two methods for the isolation of bovine herpesvirus 1 (BHV-1) from extended bovine semen

Two methods for the isolation of BHV-1 from extended bovine semen are described and their sensitivities compared. A method based on differential centrifugation is demonstrated to be superior to a standard method based on dilution when the semen extender used was milk; the two methods were equally effective when the semen extender used was egg yolk. The possible consequences of BHV-1 contamination of semen, and limitations of other isolation methods are briefly discussed.     

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. In this study we show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. Our findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation.     

Wdr5 is essential for osteoblast differentiation

Wdr5 is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro and in vivo. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs were used to stably suppress endogenous Wdr5 protein levels in MC3T3-E1 cells. Reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, evidenced by a significant decrease in alkaline phosphatase activity, Runx-2 and osteocalcin mRNAs, and absence of mineralized matrix formation. Wdr5 suppression also resulted in a reduction of histone H3 lysine 4 trimethylation, confirming its critical role in this modification. Because Wdr5 overexpression enhances canonical Wnt signaling in osteoblasts in vivo, the effects of Wdr5 silencing on this pathway were examined. The expression of the canonical Wnt target gene, c-myc, was decreased, whereas that of sfrp2, which is repressed by Wnt signaling, was increased with Wdr5 knockdown. Although only a minimal increase in apoptosis was observed, the antiapoptotic effect of Wnt signaling was also impaired with Wdr5 silencing. The expression of canonical Wnts was significantly decreased with Wdr5 knockdown, resulting in a decrease in nuclear beta-catenin protein levels. Activation of the canonical Wnt signaling pathway did not overcome the effects of Wdr5 knockdown on the expression of Wnt target genes. Chromatin immunoprecipitation demonstrated that Wdr5 is present on the Wnt1 promoter and on canonical Wnt response elements of the c-myc and Runx-2 promoters. These studies demonstrate that Wdr5 suppression interferes with the canonical Wnt signaling pathway at multiple stages and that optimal Wdr5 levels are required for induction of the osteoblast phenotype.     

Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

BackgroundSialylation of glycoproteins and glycolipids is important for biological processes such as cellular communication, cell migration and protein function. Biosynthesis of CMP-sialic acid, the essential substrate, comprises five enzymatic steps, involving ManNAc and sialic acid and their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood.Methods and resultsWe aimed to study the mechanisms of sialic acid metabolism in knockouts (KO) of the sialic acid pathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), NANS (sialic acid synthase) and CMAS (N-acylneuraminate cytidylyltransferase) genes, but was largely unaffected in NANP (N-acylneuraminate-9-phosphatase) KO, as studied by MAA and PNA lectin binding. NANP is the third enzyme in sialic acid biosynthesis and dephosphorylates sialic acid 9-phosphate to free sialic acid. LC-MS analysis of sialic acid metabolites showed that CMP-sialic acid was dramatically reduced in GNE and NANS KO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP-sialic acid levels in NANP KO were comparable to WT cells, even though sialic acid 9-phosphate, the substrate of NANP accumulated. Metabolic flux analysis with ¹³C₆-labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid.ConclusionsOur data provide evidence that NANP activity is not essential for de novo sialic acid production and point towards an alternative phosphatase activity, bypassing NANP.General significanceThis report contributes to a better understanding of sialic acid biosynthesis in humans.