Designing an artificial Golgi reactor for cell-free glycosylation

Glycosylation of therapeutically relevant proteins such as monoclonal antibodies (mAbs), is critical as it can offer increased drug efficiency, efficacy and half-life. Therefore, the production of modern biotherapeutics focuses on controlling the protein glycosylation profile using various methods. Currently, the dominating method is the traditional cell-line engineering of host cells such as mammalian cells. The main goal is to produce mAbs with a human-like glycosylation pattern. However, this approach often struggles due to high sensitivity to the fermentation environment making it difficult to scale up and control. The latter can lead to structural heterogeneity amongst the products which can be immunogenic. In addition to the in vivo methods, there are many in vitro techniques such as chemoselective or enzymatic glycosylation. However, they are often limited by the difficult implementation and, as before, product heterogeneity due to lack of control over the enzymatic reactions. In line with the need to control glycosylation in the production of therapeutic proteins, we propose an artificial Golgi reactor for in vitro glycosylation. By expressing selected glycosyltransferases and immobilizing them on solid supports we can achieve sequential enzymatic reactions required for protein glycosylation. The spatial separation will allow strict control over the reaction conditions while addressing enzyme promiscuity. Both should enhance product quality. Furthermore, we aim to perform a single-step glycosyltransferases purification/immobilization. Thanks to that, as well as the modularity of our design, the proposed system would be more sustainable and easily tailored for each application, thus producing any desired glycoform to homogeneity. A detailed mathematical approach to design and optimisation of the proposed artificial Golgi reactor focusing on mAb therapeutics has been published [1]. The authors report an optimisation of the reactor design and operational parameters that directs the whole process towards the desired glycan structure. In this research project, we have achieved expression and in vivo biotinylation of Nicotiana Tabacum GnTI (NtGnTI) and human GalT in E. coli. The biotinylated enzymes were successfully bound to streptavidin beads and used for artificial glycan synthesis. NtGnTI and GalT reacted in a sequential fashion to produce the glycan GalGlcNAcMan5GlcNAc2, as confirmed with MALDI/TOF MS analysis. In the future, we aim in extending the pathway of immobilized enzymes thus demonstrating the importance of this novel platform for in vitro glycosylation. References: [1] Klymenko, O. V., Shah, N., Kontoravdi, C., Royle, K. E. & Polizzi, K. M. Designing an Artificial Golgi reactor to achieve targeted glycosylation of monoclonal antibodies. AIChE J. 62, 2959–2973 (2016)

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation

The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-; and (iii) a nonreducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer

The Origin of Extended Disk Galaxies at z=2

Galaxy formation models typically assume that the size and rotation speed of galaxy disks are largely dictated by the mass, concentration, and spin of their surrounding dark matter haloes. Equally important, however, are the fraction of baryons in the halo that collect into the central galaxy, as well as the net angular momentum that they are able to retain during its assembly process. We explore the latter using a set of four large cosmological N-body/gasdynamical simulations drawn from the OWLS (OverWhelmingly Large Simulations) project. These runs differ only in their implementation of feedback from supernovae. We find that, when expressed as fractions of their virial values, galaxy mass and net angular momentum are tightly correlated. Galaxy mass fractions, m_d=M_gal/M_vir, depend strongly on feedback, but only weakly on halo mass or spin over the halo mass range explored here (M_vir>1e11 h^{-1}M_sun). The angular momentum of a galaxy, j_d=J_gal/J_vir, correlates with m_d in a manner that is insensitive to feedback and that deviates strongly from the simple j_d = m_d assumption often adopted in semi-analytic models of galaxy formation. The m_d-j_d correlation implies that, in a given halo, galaxy disk size is maximal when the central galaxy makes up a substantial fraction (~20%-30%) of all baryons within the virial radius. At z=2, such systems may host gaseous disks with radial scale lengths as large as those reported for star-forming disks by the SINS survey, even in moderately massive haloes of average spin. Extended disks at z=2 may thus signal the presence of systems where galaxy formation has been particularly efficient, rather than the existence of haloes with unusually high spin parameter.Comment: 5 pages, 4 figures. Accepted for publication in MNRAS Letters. Minor changes to match published versio

Does Uptake of Pharmaceuticals Vary Across Earthworm Species?

This study compared the uptake and depuration of four commonly used pharmaceuticals (carbamazepine, diclofenac, fluoxetine and orlistat) in two earthworm species (Lumbricus terrestris and Eisenia fetida). L. terrestris are a larger species and often found in deep burrows whereas E. fetida prefer to reside near the soil surface. Species burrowing habits and sizes may alter uptake by earthworms. All four pharmaceuticals were taken up into both L. terrestris and E. fetida tissue after 21 days exposure to spiked soil. Bioconcentration factors (BCFs) ranged between 1.72 and 29.83 for L. terrestris and 1.14 and 63.03 for E. fetida. For carbamazepine and diclofenac, BCFs were similar whereas for fluoxetine and orlistat, BCFs in E. fetida were more than double those seen in L. terrestris. Results indicate that uptake into earthworms cannot be generalised between species and that the influence of species traits can vary depending on the nature of the study chemical

Rates, Progenitors and Cosmic Mix of Type Ia Supernovae

Following an episode of star formation, Type Ia supernova events occur over an extended period of time, following a distribution of delay times (DDT). We critically discuss some empirically-based DDT functions that have been proposed in recent years, some favoring very early (prompt) events, other very late (tardy) ones, and therefore being mutually exclusive. We point out that in both cases the derived DDT functions are affected by dubious assumptions, and therefore there is currently no ground for claiming either a DDT strongly peaked at early times, or at late ones. Theoretical DDT functions are known to accommodate both prompt as well as late SNIa events, and can account for all available observational constraints. Recent observational evidence exists that both single degenerate and double degenerate precursors may be able of producing SNIa events. We then explore on the basis of plausible theoretical models the possible variation with cosmic time of the mix between the events produced by the two different channels, which in principle could lead to systematics effects on the SNIa properties with redshift.Comment: 9 pages, 7 figures, submitted to MNRAS March 20, accepted May 9, 2008. New version is identical to previous one apart from a "Note added in proof

Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate

As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein

The Science Case for Multi-Object Spectroscopy on the European ELT

This White Paper presents the scientific motivations for a multi-object spectrograph (MOS) on the European Extremely Large Telescope (E-ELT). The MOS case draws on all fields of contemporary astronomy, from extra-solar planets, to the study of the halo of the Milky Way and its satellites, and from resolved stellar populations in nearby galaxies out to observations of the earliest 'first-light' structures in the partially-reionised Universe. The material presented here results from thorough discussions within the community over the past four years, building on the past competitive studies to agree a common strategy toward realising a MOS capability on the E-ELT. The cases have been distilled to a set of common requirements which will be used to define the MOSAIC instrument, entailing two observational modes ('high multiplex' and 'high definition'). When combined with the unprecedented sensitivity of the E-ELT, MOSAIC will be the world's leading MOS facility. In analysing the requirements we also identify a high-multiplex MOS for the longer-term plans for the E-ELT, with an even greater multiplex (>1000 targets) to enable studies of large-scale structures in the high-redshift Universe. Following the green light for the construction of the E-ELT the MOS community, structured through the MOSAIC consortium, is eager to realise a MOS on the E-ELT as soon as possible. We argue that several of the most compelling cases for ELT science, in highly competitive areas of modern astronomy, demand such a capability. For example, MOS observations in the early stages of E-ELT operations will be essential for follow-up of sources identified by the James Webb Space Telescope (JWST). In particular, multi-object adaptive optics and accurate sky subtraction with fibres have both recently been demonstrated on sky, making fast-track development of MOSAIC feasible.Comment: Significantly expanded and updated version of previous ELT-MOS White Paper, so there is some textual overlap with arXiv:1303.002

Pathomechanistic characterization of two exonic L1CAM variants located in trans in an obligate carrier of X-linked hydrocephalus.

International audienceMutations in the gene encoding the neural cell adhesion molecule L1CAM cause several neurological disorders collectively referred to as L1 syndrome. We report here a family case of X-linked hydrocephalus in which an obligate female carrier has two exonic L1CAM missense mutations in trans substituting amino acids in the first (p.W635C) or second (p.V768I) fibronectin-type III domains. We performed various biochemical and cell biological in vitro assays to evaluate the pathogenicity of these variants. Mutant L1-W635C protein accumulates in the endoplasmic reticulum (ER), is not transported into axons, and fails to promote L1CAM-mediated cell-cell adhesion as well as neurite growth. Immunoprecipitation experiments show that L1-W635C associates with the molecular ER chaperone calnexin and is modified by poly-ubiquitination. The mutant L1-V768I protein localizes at the cell surface, is not retained in the ER, and promotes neurite growth similar to wild-type L1CAM. However, the p.V768I mutation impairs L1CAM-mediated cell-cell adhesion albeit less severe than L1-W635C. These data indicate that p.W635C is a novel loss-of-function L1 syndrome mutation. The p.V768I mutation may represent a non-pathogenic variant or a variant associated with low penetrance. The poly-ubiquitination of L1-W635C and its association with the ER chaperone calnexin provide further insights into the molecular mechanisms underlying defective cell surface trafficking of L1CAM in L1 syndrome