Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy

Oxygen-induced step bunching and faceting of Rh(553): experiment and ab initio calculations

Using a combined experimental and theoretical approach, we show that the initial oxidation of a Rh(553) surface, a surface vicinal to (111), undergoes step bunching when exposed to oxygen, forming lower-index facets. At a pressure of about 10(-6) mbar and a temperature of 380 degrees C this leads to (331) facets with one-dimensional oxide chains along the steps, coexisting with (111) facets. Further increase of the pressure and temperature results in (111) facets only, covered by an O-Rh-O surface oxide. Our density functional theory calculations provide an atomistic understanding of the observed behavior

Calculating the consequences of recovery:A European model for inhabited areas

INTRODUCTION &nbsp; The European Model for Inhabited Areas (ERMIN v1) was developed under EURANOS, an integrated project of the EC Sixth Framework Programme. It is both a model and a software tool. As a model it simulates the behaviour of radionuclides in the inhabited environment and calculates the exposure of the population as well as other relevant endpoints. As a tool it allows a user to explore different recovery options following the contamination of an urban environment with radioactive material and to refine a strategy. It has been designed to be implemented within the RODOS and ARGOS Nuclear Emergency Decision Support Systems (DSS) or as a standalone application. &nbsp; The need for a tool with the capabilities of ERMIN v1 was identified during the EC Fifth Framework programme. In particular, work during the EVATECH project indicated that decision makers recognised the need to address recovery issues by sub-dividing the area of concern on the basis of, for example, land use, deposition level and the emergency actions taken; and to explore the combined consequences of applying different packages of countermeasures within those subdivisions (Brown et al., 2004). Such a flexible approach could not be readily handled by the tools then available and it was clear that the flexibility required could only be achieved when the user was able to interact directly with a dynamic model. &nbsp; THE ERMIN V1 MODEL &nbsp; Input data required by the model include a description of the environment, initial deposition of radionuclides on to a reference surface and a description of countermeasures. ERMIN v1 can accept deposition from an atmospheric dispersion model, surface measurements and the Inhabited Area Measurement Module (IAMM) &ndash; a data assimilation module also developed under EURANOS for implementation in RODOS and ARGOS (Kaiser and Pr&ouml;hl, 2007). Countermeasures include decontamination, tie-down, shielding and relocation. &nbsp; Output information generated by the tool includes the average doses to members of the public from external exposure to gamma and beta radiation from deposited radionuclides and inhalation of resuspended radioactivity, the contamination on urban surfaces, the air concentration, the doses to workers undertaking the recovery work, the quantity and activity of waste generated and the cost and work required to implement the countermeasure. &nbsp; The model uses ratios to distribute deposition on the reference surface onto all urban surfaces. Empirical functions represent the long-term surface retention and migration in soil is simulated using a convective-dispersive model. A library of dose rates for surfaces in idealised environments is applied to calculate dose rates indoors and outdoors. Countermeasures are represented by the modification of surface contamination and the dose-rates. The ERMIN countermeasure database is based on the EUROPEAN inhabited area handbook also developed under the EURANOS project (Brown et al., 2007). &nbsp; IMPLEMENTATION AND OPERATION &nbsp; ERMIN was designed to be implemented within both the RODOS and ARGOS Nuclear Emergency DSS and also as a standalone application. Each implementation uses the same underlying executable and data library components, but the tool, as presented to the user, can be very different depending on the needs of the users of a particular system. However the basic principle of operation is the same in that the user defines regions. Within the RODOS and ARGOS DSS the regions are directly drawn onto a map (see Fig. 1), in the standalone implementation &ndash; CONDO v4 developed for UK government agencies &ndash; regions are defined in a tabular fashion. Regions may represent areas of different urban environments, different deposition (although ERMIN v1 can also accept gridded deposition from an atmospheric dispersion module), different emergency countermeasures and different packages of recovery options to be evaluated. The output in RODOS and ARGOS is generally delivered as a spatial grid overlaid onto a background map for visualisation, see Fig. 2. When using ERMIN v1, the user may make the description as complicated or as simple as necessary with regard to their immediate needs. For example, for the purposes of emergency response where a broad projection of doses without countermeasures or the evaluation of a few simple countermeasure options is required, it may be appropriate to simply define a town using a single default environment, or default mix of environments, and to assume that a single countermeasure such as road sweeping is applied everywhere. In the longer term when the emergency phase of the incident has past, it may be appropriate for the user to define different urban environments within different parts of the town and to identify different regions in which countermeasure packages can be applied

Bacterial growth on sialylated glycans.

<p>Bacterial growth on sialylated glycans.</p

Time study of trans-sialylation catalysed by Tr13.

<p>Accumulation of 3′-sialyllactose over time in 25°C, pH 3, 351 mM lactose and 8 mM cGMP-bound sialic acid.</p

Enzyme activity of Tr6 and selected mutants Tr13 and Tr6 D363E.

<p>A) Hydrolase activity on substrates pNP-Neu5Ac, 3′-sialyllactose, and cGMP. B) Trans-sialidase activity using cGMP as sialic acid donor and MU-gal as acceptor.</p

List of primers.

<p>List of primers.</p

Sequence alignment of sialidase catalytic domain from Tr6 and related trans-sialidases.

<p>Tr6 and trans-sialidases from <i>T. cruzi</i> (Uniprot ID Q26966), <i>T. congolense</i> (Uniprot ID G0WJG3) and <i>T. brucei</i> (Uniprot ID Q57XJ2) were aligned using ClustalW. Amino acids within 14 Å of sialic acid binding site are shown in bold. The seven amino acid motif is indicated with filled circles, reverting mutations are indicated with a triangle while other mutated sites are indicated with asterisks.</p