Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody drug conjugation

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro‐domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine‐scan of the mTGase pro‐domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro‐domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro‐domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30–75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site‐specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.     

Possible link between photosynthesis and leaf modulus of elasticity among vascular plants: a new player in leaf traits relationships?

A compromise between carbon assimilation and structure investment at the leaf level is broadly accepted, yet the relationship between net assimilation per area (A_n) and leaf mass per area has been elusive. We propose bulk modulus of elasticity (ε) as a suitable parameter to reflect both leaf structure and function, and an inverse relationship between ε and A_n and mesophyll conductance (g_m) is postulated. Using data for A_n, g_m and ε from previous studies and new measurements on a set of 20 species covering all major growth forms, a negative relationship between A_n or g_m and ε was observed. High ε was also related to low leaf capacitance and higher diffusive limitations to photosynthesis. In conclusion, ε emerges as a key trait linked with photosynthetic capacity across vascular plants, and its relationship with g_m suggests the existence of a common mechanistic basis, probably involving a key role of cell walls.     

DNA barcodes,cryptic diversity and phylogeography of a W Mediterranean assemblage of thermosbaenacean crustaceans

We assess the occurrence of crypticism and analyse the phylogeography of a thermosbaenacean crustacean, the monodellid Tethysbaena scabra, endemic to the Balearic Islands (W Mediterranean). This species occurs only in mixohaline waters of coastal wells and caves adjacent to the seashore. We have used the mitochondrial DNA barcode region to assess its genetic population structure throughout the anchialine environment of the islands. Maximum likelihood phylogenetic analyses showed that the Balearic Tethysbaena and those from the NW Italian Peninsula form a monophyletic assemblage subdivided into several lineages. Cytochrome c oxidase subunit 1 (cox1) p‐distances among the more divergent Mallorcan lineages are remarkably high and on par with those established between the formally described species T. scabra from Menorca and T. argentarii from Italy. This result and the application of the generalised mixed Yule coalescence model (GMYC) suggest that at least some of the Mallorcan lineages represent cryptic species. A clear‐cut phylogeographic pattern is displayed by this anchialine assemblage: six of its seven lineages appear in allopatry, with the exception of a Mallorcan lineage limited to a single cave nested within the geographic range of another lineage. All lineages show a distribution reduced to a single cave or to short portions of coast not exceeding 60 km in length. Our coalescence estimations suggest an early Tortonian (10.7 Ma) origin for the Balearic + Italy Tethysbaena clade, an age that is largely prior to the onset of the eustatic oscillations associated with the Quaternary glaciations. Only the diversification that took place within some of the Mallorcan lineages could be coeval with the broad glacio‐eustatic oscillations of the Quaternary.     

Model for Probing Membrane-Cortex Adhesion by Micropipette Aspiration and Fluctuation Spectroscopy

We propose a model for membrane-cortex adhesion that couples membrane deformations, hydrodynamics, and kinetics of membrane-cortex ligands. In its simplest form, the model gives explicit predictions for the critical pressure for membrane detachment and for the value of adhesion energy. We show that these quantities exhibit a significant dependence on the active acto-myosin stresses. The model provides a simple framework to access quantitative information on cortical activity by means of micropipette experiments. We also extend the model to incorporate fluctuations and show that detailed information on the stability of membrane-cortex coupling can be obtained by a combination of micropipette aspiration and fluctuation spectroscopy measurements.     

A conserved tetrameric interaction of cry toxin helix α3 suggests a functional role for toxin oligomerization

Crystal (Cry) toxins are widely used for insect control, but their mechanism of toxicity is still uncertain. These toxins can form lytic pores in vitro, and water soluble tetrameric pre-pore intermediates have been reported. Even the precise oligomeric state of the toxin in membranes, trimeric or tetrameric, is still a debated issue. Based on previous reports, we have assumed that interactions between toxin monomers in solution are at least partly mediated by domain I, and we have analyzed in silico the homo-oligomerization tendencies of the domain I α-helices individually. Using many homologous sequences for each α-helix, our strategy allows selection of evolutionarily conserved interactions. These interactions appeared only in helices α3 and α5, but only α3 produced a suitably oriented or α-helical sample in lipid bilayers, forming homotetramers in C14-betaine, and allowing determination of its rotational orientation in lipid bilayers using site-specific infrared dichroism (SSID). The determined orientation in the tetrameric model is in agreement with only one of the evolutionarily conserved models. In addition mutation R99E, which was found to inhibit oligomerization experimentally, greatly destabilized the tetramer in molecular dynamic simulations. In this model, helix 3 is able to form inter-monomer interactions without significant rearrangements of domain I, which is compatible with the available crystal structure of Cry toxins in solution. The model presented here at least partially explains the reported tetrameric oligomerization of Cry toxins in solution and the inhibition of this oligomerization by a synthetic α3 peptide.     

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.       

Effect of nutrition and Enteromyxum leei infection on gilthead sea bream Sparus aurata intestinal carbohydrate distribution

The effect of a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source on the mucosal carbohydrate pattern of the intestine was studied in gilthead sea bream Sparus aurata challenged with the myxosporean parasite Enteromyxum leei. Fish fed for 9 mo either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) were exposed to parasite-contaminated water effluent. Samples of the anterior, middle and posterior intestine (AI, MI and PI, respectively) were obtained for parasite diagnosis and histochemistry. Fish were categorised as control (C, not exposed), early (E) or late (L) infected. Mucin and lectin histochemistry was applied to detect the different types of mucins and sialic acid in goblet cells (GC), the brush border and enterocytes. The number of GC stained with periodic acid Schiff (PAS), alcian blue (AB), aldehyde fuchsin-alcian blue (AF-AB), for the detection of neutral, acidic, sulphated and carboxylic mucins, and with the lectin Sambucus nigra agglutinin (SNA), were counted in digital images. The 66VO diet produced a significant decrease of GC with neutral and acidic mucins in the AI and MI, and also of those with carboxylic mucins and sialic acid in the MI. Sulphated mucins and sialic acid were less abundant in the AI than in the MI and PI in the C-66VO treatment. E. leei infection had a strong effect on the number of GC, as E and L infected fish had a significant decrease of GC positive for all the stains versus C fish in PI. Time and diet effects were also observed, since the lowest values were mostly registered in E-66VO fish in PI. In conclusion, though GC depletion was mainly induced by enteromyxosis, an effect of the diet was also observed. Thus, the diet can be a predisposing factor that worsens the disease course.