Olivier Roitel

Effect of surfactant pluronic F‐68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN‐γ in mild operating conditions

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N‐glycosylation sites, γ‐interferon (IFN‐γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F‐68 (PF‐68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN‐γ produced by a CHO cell line that was maintained in a rich protein‐free medium in the absence or presence of low agitation. Under these conditions, a dose‐dependent effect of PF‐68 (0–0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF‐68 led to increased IFN‐γ production as a result of both higher cell densities and a higher specific production rate of IFN‐γ. If cells were grown with agitation, lack of PF‐68 in the culture medium decreased the fraction of the fully glycosylated IFN‐γ glycoform (2N) from 80% to 65–70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF‐68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.

Lipolysis stimulated lipoprotein receptor: a novel molecular link between hyperlipidemia, weight gain, and atherosclerosis in mice.

The lipolysis-stimulated lipoprotein receptor, LSR, is a multimeric protein complex in the liver that undergoes conformational changes upon binding of free fatty acids, thereby revealing a binding site (s) that recognizes both apoB and apoE. Complete inactivation of the LSR gene is embryonic lethal in mice. Here we show that removal of a single LSR allele (LSR-/+) caused statistically significant increases in both plasma triglyceride and cholesterol levels, a 2-fold increase in plasma triglyceride changes during the post-prandial phase, and delayed clearance of lipid emulsions or a high fat meal. The longer postprandial lipoprotein clearance time observed in LSR-/+ mice was further increased in LSR-/+ mice lacking functional low density lipoprotein (LDL) receptors. LSR-/+ mice placed on a Western-type diet displayed higher plasma triglycerides and cholesterol levels, increased triglyceride-rich lipoproteins and LDL, and increased aorta lipid content, as compared with control mice on the same diet. Furthermore, a direct correlation was observed between the hyperlipidemia and weight gain but only in the LSR-/+ mice. Knockdown of LSR expression by small interfering RNA in mouse Hepa1-6 cells led to decreased internalization of both DiI-labeled cyclohexanedione-LDL and very low density lipoprotein in the presence of oleate. These data led us to conclude that LSR contributes to the physiological clearance of atherogenic triglyceride-rich lipoproteins and LDL. We propose that LSR cooperates with the LDL receptor in the final hepatic processing of apoB-containing lipoproteins and represents a novel therapeutic target for the treatment of hyperlipidemia associated with obesity and atherosclerosis.

The crystal structure of the FAD/NADPH binding domain of flavocytochrome P450 BM3

We report the crystal structure of the FAD/NADPH‐binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP+, identifying important protein interactions with the NADPH 2′‐phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH‐dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild‐type FAD domain to be near‐exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme’s connecting domain (that links the FAD domain to the FMN‐binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin‐like) domain within the BM3 reductase module.

Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

Detection of IgE-reactive proteins in hydrolysed dog foods

BACKGROUND Commercial hydrolysed diets are used for the diagnosis of food allergy in dogs. The cleaved parent proteins are presumed to be too small to elicit an allergic response by reacting with allergen-specific immunoglobin E (IgE). OBJECTIVES To evaluate three commercial hydrolysed dog diets for proteins. ANIMALS Sera were collected from dogs with suspected food allergy. METHODS Two batches of each hydrolysed diet were examined by electrophoresis and visualized by Coomassie blue, silver nitrate staining and IgE immunoblotting. RESULTS From two to five proteins, ranging from 21 to 67 kDa, were detected in all three diets evaluated. Circulating IgE antibodies targeting these proteins were detected by immunoblotting of dog sera. Six different carbohydrate proteins were identified by mass spectrometry; maize/potato granule-bound starch synthase-1, soybean glycinin, soybean β-conglycinin α chain, potato aspartic protease inhibitor, rice glutelin type B1 and soybean sucrose-binding protein. Four of these proteins have been described as allergens in humans. CONCLUSIONS Some commercial hydrolysed diets contain carbohydrate proteins. Some dogs have circulating IgE antibodies targeting these proteins. The clinical significance of these findings is unknown.

Flavocytochrome P450 BM3: An update on structure and mechanism of a biotechnologically important enzyme

Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.

Abstract 5287: RNA DNA divergences: An unsuspected marker of cancer genomic instability accurately predicts triple-negative breast cancer severity

Next generation sequencing provides 3 measures of cancer genomic instability i.e. somatic DNA variations, differential gene expression and RNA DNA divergences (RDD). The latter reflects changes in RNA sequences that are not present at the DNA level. Triple negative breast cancers (TNBC) represent the most severe form of the disease and are currently not amenable to targeted therapies nor to prognosis testing. We used the 3 measures of genomic instability to construct specific optimal algorithms that effectively separated 20 TNBC patients with poor or good clinical outcomes: 11 patients died from the disease within 1000 days following diagnosis of non metastatic TNBC while 9 were alive after 2500 days of follow up. All 3 models efficiently separated these 2 clinically polarized groups. However, only RDD based algorithms and not those relying on somatic mutations and expression profiles retains performances in excess of 90% accuracy after statistical cross validation. The 3 models were then applied in blind to 45 unknown patients with the same inclusion criteria i.e. non metastatic TNBC diagnosed before the age of 65 irrespective of menopausal, lymph node, tumor size and ethnic origin or recruitment centers. Kaplan-Meier analysis showed that the RDD based algorithm was highly predictive of clinical outcome i.e. 100% of patients predicted with good outcome were alive while 80% of patients predicted with poor outcome died in the same time interval (p Citation Format: Bernard E. Bihain, Stephane Verdun, Julie Tomasina, Benoit Hilselberger, Marie Brulliard, Lionel Bonnard, Marina Trarbach, Olivier Roitel, Sandrine Jacquenet, Virginie Ogier, Jean-Pierre Armand, Benoit Thouvenot. RNA DNA divergences: An unsuspected marker of cancer genomic instability accurately predicts triple-negative breast cancer severity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5287. doi:10.1158/1538-7445.AM2015-5287

Improving diagnostic performances of peanut allergy using novel molecular components

Accurate in-vitro component-based diagnosis will allow clinicians to focus their efforts toward complex issues such as disease severity and reactive dose evaluation as well as establishing effective eviction measures. However to achieve this, clinical utility of component-resolved peanut allergy testing must be rigorously evaluated. In this pilot study, we compared performances of 2 component-resolved peanut allergy assays from Hycor® [H] and Thermo Scientific® [T] that are based on recombinant allergens (rAra h 1 [H/T], rAra h 2 [H/T], rAra h 3 [H/T], rAra h 6 [H], rAra h 7 [H], rAra h 8 [H/T] and rAra h 9 [H/T]). IgE reactivity to each allergen was measured in 34 clinically characterized patients: 26 peanut-allergic and 8 atopic subjects from Eastern and Northern France. Total peanut extract assay [T] yielded low performances even with a 0.35 kU/L cut-off (sensitivity: 92%, specificity: 25%). Quantifications of IgE to rAra h 1, rAra h 2 and rAra h 3 show a strong correlation between the two manufacturers (rAra h 1: r2 = 0.85, p < 10-3 ; rAra h 2: r2 = 0.96, p < 10-3; rAra h 3: r2 = 0.90, p < 10-3) and achieve the same level of performances independently of the allergen source. Among these three assays, rAra h 2 [H/T] yielded a sensitivity of 85% and a specificity of 100% with a 0.35 kU/L cut-off. The rAra h 6 assay is available from Hycor® only and yielded a sensitivity of 92% and a specificity of 100% using the same cut-off, i.e. 24/26 peanut allergic patients are positive for rAra h 6. The IgE from the 2 undiagnosed patients do not react with any of the other recombinant from any sources. Neither Hycor® nor Thermo Scientific® rAra h 8 assays are clinically useful for peanut allergy diagnosis in spite of their specificity differences at 50% and 25% respectively. Thermo Scientific® rAra h 9 assay was not clinically useful with no positive sample detected. Hycor® rAra h 9 assay detected 2 of the 26 peanut allergic patients without reactivity in the atopic patients (specificity: 12%, sensitivity: 100%). This is consistent with the low prevalence of Ara h 9 sensitisation in Northern Europe. This study suggests the possibility of improving the clinical utility of molecular component assays by substituting rAra h 6 to rAra h 2. It also introduces rAra h 9 as a novel component that might be a significant clinical value in countries outside Northern Europe. This will be evaluated in multicenter studies.

Flavocytochrome P450 BM3: an update on structure and mechanism of a biotechnologically important enzyme.

Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.