Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Background

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/Principal Findings

We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions

We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.     

6H,13H-Pyrazino[1,2-a;4,5-a']diindole analogs: probing the pharmacophore for allosteric ligands of muscarinic M2 receptors

A series of 6H,13H-pyrazino[1,2-a;4,5-a']diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M(2) receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [(3)H]N-methylscopolamine-occupied M(2) receptors (reflected by EC(50,diss)) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand-receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by K(i)) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M(2) receptors.     

Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition

The RNA-dependent RNA polymerase (NS5B) from hepatitis C virus (HCV) is a key enzyme in HCV replication. NS5B is a major target for the development of antiviral compounds directed against HCV. Here we present the structures of three thiophene-based non-nucleoside inhibitors (NNIs) bound non-covalently to NS5B. Each of the inhibitors binds to NS5B non-competitively to a common binding site in the "thumb" domain that is approximately 35 Angstroms from the polymerase active site located in the "palm" domain. The three compounds exhibit IC(50) values in the range of 270 nM to 307 nM and have common binding features that result in relatively large conformational changes of residues that interact directly with the inhibitors as well as for other residues adjacent to the binding site. Detailed comparisons of the unbound NS5B structure with those having the bound inhibitors present show that residues Pro495 to Arg505 (the N terminus of the "T" helix) exhibit some of the largest changes. It has been reported that Pro495, Pro496, Val499 and Arg503 are part of the guanosine triphosphate (GTP) specific allosteric binding site located in close proximity to our binding site. It has also been reported that the introduction of mutations to key residues in this region (i.e. Val499Gly) ablate in vivo sub-genomic HCV RNA replication. The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.     

Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding.     

Determinants for Membrane Association of the Hepatitis C Virus NS2 Protease Domain

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is required for HCV polyprotein processing and particle assembly. It comprises an N-terminal membrane domain and a C-terminal, cytosolically oriented protease domain. Here, we demonstrate that the NS2 protease domain itself associates with cellular membranes. A single charged residue in the second α-helix of the NS2 protease domain is required for proper membrane association, NS2 protein stability, and efficient HCV polyprotein processing.     

Impact of the zebra mussel Dreissena polymorpha invasion on the budget of suspended material in a shallow lagoon ecosystem

The role of the zebra mussel Dreissena polymorpha in redistribution of total particulate material (TPM) between the water column and bottom sediment was estimated using the TPM budget for a mussel bed in the Curonian lagoon, the Baltic Sea. Seasonal clearance rates were derived from the TPM budget assuming two resuspension scenarios: no resuspension and full resuspension of biodeposits. Estimated clearance rates for both scenarios were compared with the rates calculated from the population clearance rate model. Seasonal clearance rates estimated using the population model (1.1 and 11.8 l g⁻¹ SFDW day⁻¹) fitted well into the interval of seasonal clearance rates calculated from TPM budgets assuming no resuspension of biodeposits (3.2 and 21.4 l g SFDW⁻¹ day⁻¹). In the scenario with biodeposits resuspension clearance rates were much higher (57.4 and 148.9 g SFDW⁻¹ day⁻¹). The ratio of clearance to residence time was highly dependent on the fate of biodeposits. Therefore its use in interpretation of the species impact on TPM was limited. An alternative measure based on the ratio of the amount of TPM biodeposited to TPM transported into the bed was used. It was found that zebra mussels are able to deposit between 10 and 30% of the incoming TPM, and the amount of biodeposited material was correlated with water residence time. Results indicate that the impact of zebra mussels on TPM in the lagoon is small relative to the high transport rates of TPM over the bed. However, annual biosedimentation rate (~590 g m⁻²) in the mussel bed was higher than physical deposition rate (~380 g m⁻²) in accumulation areas devoid of large suspension feeders. We suggest that a local impact due to enhanced availability of organic material to other trophic groups of associated benthic organisms may be more significant than effects on TPM pathways at an ecosystem scale.     

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.     

Validated method for the therapeutic drug monitoring of flunitrazepam in human serum using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry with an ion trap detector

An HPLC–MS–MS method for the quantitative analysis of flunitrazepam in human serum was established. The method features a very simple liquid–liquid extraction, the use of a standard 4-mm HPLC column, isocratic elution using a buffer-free solvent, short retention times in connection with good peak resolution and the sensitivity and selectivity of an ion trap MS–MS detector. The procedure enables unambiguous identification of analytes by their product ion spectra, as well as sensitive quantitation (limit of quantitation for flunitrazepam=0.5 ng/ml). This feature was used for the confirmation of HPLC–UV results for nitrazepam.