Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135–1148, 2016     

Metabolomics reveals dose effects of low-dose chronic exposure to uranium in rats: identification of candidate biomarkers in urine samples

Introduction

Data are sparse about the potential health risks of chronic low-dose contamination of humans by uranium (natural or anthropogenic) in drinking water. Previous studies report some molecular imbalances but no clinical signs due to uranium intake.

Objectives

In a proof-of-principle study, we reported that metabolomics is an appropriate method for addressing this chronic low-dose exposure in a rat model (uranium dose: 40 mg L^?1; duration: 9 months, n = 10). In the present study, our aim was to investigate the dose–effect pattern and identify additional potential biomarkers in urine samples.

Methods

Compared to our previous protocol, we doubled the number of rats per group (n = 20), added additional sampling time points (3 and 6 months) and included several lower doses of natural uranium (doses used: 40, 1.5, 0.15 and 0.015 mg L^?1). LC–MS metabolomics was performed on urine samples and statistical analyses were made with SIMCA-P+ and R packages.

Results

The data confirmed our previous results and showed that discrimination was both dose and time related. Uranium exposure was revealed in rats contaminated for 9 months at a dose as low as 0.15 mg L^?1. Eleven features, including the confidently identified N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide and 4-hydroxyphenylacetylglycine, discriminated control from contaminated rats with a specificity and a sensitivity ranging from 83 to 96 %, when combined into a composite score.

Conclusion

These findings show promise for the elucidation of underlying radiotoxicologic mechanisms and the design of a diagnostic test to assess exposure in urine, in a dose range experimentally estimated to be above a threshold between 0.015 and 0.15 mg L^?1.

     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.     

Insights into the Function of YciM,a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.     

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.     

A metapopulation model for the spread and persistence of contagious bovine pleuropneumonia (CBPP) in African sedentary mixed crop-livestock systems

Contagious bovine pleuropneumonia (CBPP) is endemic in several developing countries. Our objective is to evaluate the regional CBPP spread and persistence in a mixed crop-livestock system in Africa. A stochastic compartmental model in metapopulation is used, in which between-herd animal movements and the within-herd infection dynamics are explicitly represented. Hundred herds of varying size are modelled, each sending animals to n other herds (network degree). Animals are susceptible, latent, infectious, chronic carrier or resistant. The role of chronic carriers in CBPP spread being still debated, several chronic periods and infectiousness are tested. A sensitivity analysis is performed to evaluate the influence on model outputs of these parameters and of pathogen virulence, between-herd movement rate, network degree, and calves recruitment. Model outputs are the probability that individual- and group-level reproductive numbers R₀ and R_* are above one, the metapopulation infection duration, the probability of CBPP endemicity (when CBPP persists over 5 years), and the epidemic size in infected herds and infected animals. The most influential parameters are related to chronic carriers (infectiousness and chronic period), pathogen virulence, and recruitment rate. When assuming no CBPP re-introduction in the region, endemicity is only probable if chronic carriers are assumed infectious for at least 1 year and to shed the pathogen in not too low an amount. It becomes highly probable when assuming high pathogen virulence and high recruitment rate.     

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase,Functionally Equivalent to the Class III Fbp Enzyme

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.Under glycolytic growth conditions, unidirectional phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate is catalyzed by the 6-phosphofructokinase (EC 2.7.1.11). Under gluconeogenic growth conditions, the opposite reaction is catalyzed by the fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) and is required for the synthesis of fructose-6-phosphate and derived metabolites, such as cell wall precursors. Escherichia coli possesses two FBPases: the class I FBPase, encoded by fbp, is highly similar to eukaryotic enzymes, and the class II FBPase (GlpX) (3) has homologues in nearly all prokaryotic genera but in only a few eukaryotes (a green alga, an amoeba, and a moss) and a few archaean species (of the Methanosarcina genus). Biochemical, physiological, and genetic studies allowed the characterization of a Bacillus subtilis enzyme which defined a new class of bacterial FBPases (class III) not structurally related to those previously described and found mainly in Firmicutes (5-7). The gene encoding this activity was identified and, although structurally unrelated to the E. coli class I FBPase gene, was also named fbp (8). In E. coli, the major FBPase is the class I Fbp, whereas the class II GlpX seems to play a minor role (3). In other organisms, the major or even the only FBPase belongs to the class II GlpX family: Bacillus cereus possesses two glpX-like genes and no class I or class III FBPase-encoding gene (26); in Mycobacterium tuberculosis, FBPase activity is encoded only by a glpX-like gene, which has been shown to complement an E. coli mutant lacking such activity (18); in Corynebacterium glutamicum, the only FBPase, essential for growth on gluconeogenic carbon sources, belongs to class II (19). It has been shown that a B. subtilis fbp mutant was still able to grow on substrates such as d-fructose, glycerol, or l-malate as the sole carbon source, which indicated that this mutant could bypass the FBPase reaction during gluconeogenesis (6). Random mutagenesis (ethyl methanesulfonate treatment) performed with this fbp mutant enabled the definition of a B. subtilis locus (bfd) whose additional mutation prevented growth on gluconeogenic carbon sources, but this locus had not been characterized further (7). Determination of the nucleotide sequence of the whole B. subtilis chromosome (16) led to the identification of a putative gene, ywjI, encoding a protein displaying strong homologies with GlpX family members (e.g., 54% identity and 74% similarity with GlpX from C. glutamicum). This gene has therefore been annotated glpX, encoding a class II FBPase, but such annotation has never been validated by genetic or biochemical experimental evidence. In this work, we present experimental evidence that ywjI indeed encodes a class II FBPase.