Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Associations between Extending Access to Primary Care and Emergency Department Visits: A Difference-In-Differences Analysis

BackgroundHealth services across the world increasingly face pressures on the use of expensive hospital services. Better organisation and delivery of primary care has the potential to manage demand and reduce costs for hospital services, but routine primary care services are not open during evenings and weekends.Extended access (evening and weekend opening) is hypothesized to reduce pressure on hospital services from emergency department visits. However, the existing evidence-base is weak, largely focused on emergency out-of-hours services, and analysed using a before-and after-methodology without effective comparators.ConclusionsThe study found that extending access was associated with a reduction in emergency department visits in the first 12 months. The results of the research have already informed the decision by National Health Service England to extend primary care access across Greater Manchester from 2016. However, further evidence is needed to understand whether extending primary care access is cost-effective and sustainable.     

Predicting ratings of perceived exertion in youth soccer using decision tree models

The purpose of this study was to determine the effectiveness of white-box decision tree models (DTM) for predicting the rating of perceived exertion (RPE). The second aim was to examine the relationship between RPE and external measures of intensity in youth soccer training at the group and individual level. Training load data from 18 youth soccer players were collected during an in-season competition period. A total of 804 training observations were undertaken, with a total of 43 ± 17 sessions per player (range 12–76). External measures of intensity were determined using a 10 Hz GPS and included total distance (TD, m/min), high-speed running distance (HSR, m/min), PlayerLoad (PL, n/min), impacts (n/min), distance in acceleration/deceleration (TD ACC/TD DEC, m/min) and the number of accelerations/decelerations (ACC/DEC, n/min). Data were analysed with decision tree models. Global and individualized models were constructed. Aggregated importance revealed HSR as the strongest predictor of RPE with relative importance of 0.61. HSR was the most important factor in predicting RPE for half of the players. The prediction error (root mean square error [RMSE] 0.755 ± 0.014) for the individualized models was lower compared to the population model (RMSE 1.621 ± 0.001). The findings demonstrate that individual models should be used for the assessment of players’ response to external load. Furthermore, the study demonstrates that DTM provide straightforward interpretation, with the possibility of visualization. This method can be used to prescribe daily training loads on the basis of predicted, desired player responses (exertion).     

Characterization of excretory/secretory endopeptidase and metallo-aminopeptidases from Taenia crassiceps metacestodes

Cysticercosis is caused by Taenia spp. metacestodes, which must survive in the host tissues to complete their life cycle. Their survival depends on their control of host immune responses. Because many parasites use proteases to modulate host responses, we examined culture media from Taenia crassiceps metacestodes for protease activity using peptide substrates. We identified prominent aminopeptidase activity at neutral pH, which was inhibited by chelating agents and partially inhibited by the aminopeptidase inhibitor, bestatin. Endopeptidase substrates were optimally cleaved at slightly acidic pH and endopeptidase activity was inhibited by cysteine protease inhibitors. Gel filtration FPLC and subsequent visualization by silver staining revealed a metallo-aminopeptidase of molecular weight 21 kDa and cysteine proteases of Mr 70 and 64 kDA. Recombinant IL-2 was digested when incubated with parasite culture supernatants, but not with control media. IL-2 degradation was completely inhibited by 1,10 phenanthroline and partially inhibited by bestatin, suggesting that a metallo-aminopeptidase was responsible. Incubation of human IgG with culture supernatants resulted in complete degradation of IgG, which was blocked by cysteine protease inhibitors. These observations demonstrate that Taenia spp. metacestodes secrete a number of proteolytic enzymes, which may target molecules from the host immune system and assist in evasion of the host immune response.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.The structure of many proteins can be altered by post-translational modifications (1). Although the impact of post-translational modifications (PTMs)¹ on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2), it is less defined for other PTMs and in many cases is protein-dependent. Because there are many important downstream effects of PTMs, including changes in protein localization, protein and cellular diversification, protein functionality, protein stability, protein life cycle, and so forth, understanding how PTMs alter protein structure for as many proteins as possible in a timely manner is a highly desirable goal. Furthermore, in an age where recombinant proteins are being used to treat disease, it becomes ever more important to understand how particular modifications may alter the structure and eventually the function of therapeutic proteins. To realize these goals, methods that permit access to conformational information for modified forms of therapeutic proteins must be developed and refined. In this report, we will illustrate how MS can contribute to structural proteomics by describing our recent work with a recombinant monoclonal antibody (an IgG1), which represents an important class of therapeutic proteins.Many biopharmaceutical companies are pursuing antibody drugs (3). In particular, the IgG1 subclass of antibodies has evolved into a commonly used therapeutic option for the treatment of a wide range of diseases. IgG1s consist of a dimer of identical heavy chains and light chains that fold to form (from N to C terminus) the variable, CL, CH1, CH2, and CH3 domains (as an example, see Ref. 4). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5). The variable, CL, and CH1 domains are collectively referred to as the Fab (fragment antigen binding) portion of IgG1, which is responsible for recognizing a specific antigen. The CH2 and CH3 domains together are referred to as the Fc (fragment crystallizable) portion, which carries out effector functions such as binding to Fcγ receptors. These effector functions are essential to many therapeutic antibodies, especially when antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity are involved in the mechanisms of action (6).As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood at a functional level. In the case of IgG1s, with over 1300 amino acid residues and a molecular mass approaching 150 kDa, a large array of PTMs can be incorporated both in vivo (during cellular synthesis) and in vitro (as a result of handling and processing steps that occur during purification, vialing, and storage). Commonly monitored PTMs on IgG1s include methionine oxidation, asparagine and glutamine deamidation, N-terminal acetylation or cyclization, glycation of lysine, and variable glycosylation (8). Some of these modifications affect only a small percentage of the protein product, and their presence may not change overall outcome. Others, however, can have significant impact on the structure, function, and biological activities of a protein that can involve self-association as well as interactions with other proteins (9). The same PTMs can affect different IgG1 molecules in different ways or have no effect(s) at all. Therefore assessing the presence of PTMs, determining the relative level of the modifications, and understanding the structural effects of PTMs are all important during development of protein biopharmaceuticals.Two commonly studied IgG1 modifications are methionine oxidation and glycosylation, each of which has been shown to affect biological function (6, 10). Methionine oxidation has been implicated in protein stability (inducing aggregation), and increased oxidation levels have been shown to provoke an immunogenic response (11–13). Elevated levels of methionine oxidation in an IgG1 were shown to impact neonatal Fc receptor (FcRn) and protein A binding (10). Variable glycosylation (i.e. different levels of sialic acid, galactose, fucose, or high mannose structures) is known to influence thermal stability and effector functions (14–16). Previous studies have shown that removal of fucose from the glycan present on the Fc portion of an IgG1 can greatly enhance Fc binding to FcγRIIIa, but removal of the entire glycan nearly abolishes FcγRIIIa binding (17). As oxidation and changes to the glycan are both common IgG1 modifications, we were interested in determining the conformational effects of oxidation, afucosylation, and galactosylation and correlating any conformational changes that were observed with changes of FcγRIIIa binding activity.Conformational analysis of large proteins like antibodies, however, is not trivial. Traditional biophysical techniques such as circular dichroism, DSC, and fluorescence provide useful information, but these techniques look at the entire protein and provide only a global view (18). NMR and x-ray crystallography can both provide high resolution structural analysis, but each is faced with limitations that often make the study of an intact IgG1 difficult or nearly impossible (19–21). Recently we described how hydrogen/deuterium exchange (H/DX) MS could be used to study the conformation and conformational dynamics of an intact IgG1 with resolution down to stretches of several amino acid residues (22). For the present work, we used H/DX MS to study the impact of galactosylation, oxidation, and afucosylation on the conformation and dynamics of an intact IgG1. We also studied the complex of IgG1 and FcγRIIIa to map the points of interaction and probe any changes in the dynamics of the IgG1 as a result of FcγRIIIa interaction. Finally, we correlated the functional activity of all the proteins that were studied by H/DX MS with the observed conformational disturbance(s). Such correlations are important to connect structure with function and to understand whether a particular PTM is something that may affect the therapeutic value of a recombinant protein.