A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Dislocation of Type I Membrane Proteins from the ER to the Cytosol Is Sensitive to Changes in Redox Potential

The human cytomegalovirus (HCMV) gene products US2 and US11 dislocate major histocompatibility class I heavy chains from the ER and target them for proteasomal degradation in the cytosol. The dislocation reaction is inhibited by agents that affect intracellular redox potential and/or free thiol status, such as diamide and N-ethylmaleimide. Subcellular fractionation experiments indicate that this inhibition occurs at the stage of discharge from the ER into the cytosol. The T cell receptor α (TCR α) chain is also degraded by a similar set of reactions, yet in a manner independent of virally encoded gene products. Diamide and N-ethylmaleimide likewise inhibit the dislocation of the full-length TCR α chain from the ER, as well as a truncated, mutant version of TCR α chain that lacks cysteine residues. Cytosolic destruction of glycosylated, ER-resident type I membrane proteins, therefore, requires maintenance of a proper redox potential for the initial step of removal of the substrate from the ER environment.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.     

A duplex real-time PCR assay for detection of drug-induced mitochondrial DNA depletion in HepG2 cells

Screening of drug-induced mitochondrial DNA (mtDNA) depletion during early preclinical drug development is of major interest. Here we describe the establishment of a novel duplex calibrator-normalized real-time polymerase chain reaction (PCR) assay for rapid and reliable quantification of mtDNA in HepG2 cells. This assay involves quantification of an mtDNA target gene (cytochrome b) relative to a nuclear DNA (nDNA) reference gene (β-actin) in one tube. The assay was evaluated for its precision, linearity, and reproducibility, and reliable detection of mtDNA depletion was demonstrated. Using this novel real-time PCR assay, drug-induced mtDNA depletion could be accurately detected.     

EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.     

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.     

The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.