Improving biologics development by high performance glycoanalysis

Glycomics is a rapidly emerging field that can be viewed as a complement to other „omics“ approaches including proteomics and genomics. Hence, there is a dramatic increase in the demand for analytical tools and specific databases in glycobiology, respectively, glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, fully automated, highly sensitive, reliable, high-throughput and high-resolution analysis methods including automated data evaluation are required. One very promising method is based on multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (xCGE-LIF). The glycoanalytical approach established includes sample preparation and measuring methods, software, and database solutions to tackle challenges in a great number of application fields. First, an optimized modular sample preparation workflow is presented with respect to performance and feasibility regarding high-throughput analytics [1-5]. Second, parallel sample-measurement is shown to result in massive reduction of the effective run-time per sample [4]. Third, automated data analysis with a newly developed modular software-tool for data processing and data analysis is demonstrated that involves integration of a corresponding oligosaccharide-database [6-8]. Using this high-performance xCGE-LIF based glycoanalysis system, the generated “normalized” electropherograms of glycomoieties (“fingerprints”) can be evaluated on three levels: (1) “simple” qualitative and quantitative pattern comparison (“fingerprinting”), (2) identification of compounds in complex mixtures via database matching (“glycoprofiling”) and (3) extended structural analysis using exoglycosidase sequencing in combination with xCGE-LIF based glycoprofiling. The broad applicability of the system is demonstrated for different types of glycosamples: from manufacturing of biologics and vaccines (including recombinant and viral glycoproteins) [1-3], to human stem cells, blood serum [4,5] and milk [8]

DeNovoGUI: an open source graphical user interface for de novo sequencing of tandem mass spectra

De novo sequencing is a popular technique in proteomics for identifying peptides from tandem mass spectra without having to rely on a protein sequence database. Despite the strong potential of de novo sequencing algorithms, their adoption threshold remains quite high. We here present a user-friendly and lightweight graphical user interface called DeNovoGUI for running parallelized versions of the freely available de novo sequencing software PepNovo+, greatly simplifying the use of de novo sequencing in proteomics. Our platform-independent software is freely available under the permissible Apache2 open source license. Source code, binaries, and additional documentation are available at http://denovogui.googlecode.com.acceptedVersio

Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3

Hydroquinone-1,2-dioxygenase, an enzyme involved in the degradation of alkylphenols in Sphingomonas sp. strain TTNP3 was purified to apparent homogeneity. The extradiol dioxygenase catalyzed the ring fission of hydroquinone to 4-hydroxymuconic semialdehyde and the degradation of chlorinated and several alkylated hydroquinones. The activity of 1 mg of the purified enzyme with unsubstituted hydroquinone was 6.1 μmol per minute, the apparent Km 2.2 μM. ICP-MS analysis revealed an iron content of 1.4 moles per mole enzyme. The enzyme lost activity upon exposure to oxygen, but could be reactivated by Fe(II) in presence of ascorbate. SDS-PAGE analysis of the purified enzyme yielded two bands of an apparent size of 38 kDa and 19 kDa, respectively. Data from MALDI-TOF analyses of peptides of the respective bands matched with the deduced amino acid sequences of two neighboring open reading frames found in genomic DNA of Sphingomonas sp strain TTNP3. The deduced amino acid sequences showed 62% and 47% identity to the large and small subunit of hydroquinone dioxygenase from Pseudomonas fluorescens strain ACB, respectively. This heterotetrameric enzyme is the first of its kind found in a strain of the genus Sphingomonas sensu latu

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo

FUT6 deficiency compromises basophil function by selectively abrogating their sialyl-Lewis x expression.

Sialyl-Lewis x (sLex, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLex expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLex+ and sLex- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis