[Show abstract][Hide abstract] ABSTRACT: Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.
[Show abstract][Hide abstract] ABSTRACT: We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.
[Show abstract][Hide abstract] ABSTRACT: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting.
Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein.
Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.
[Show abstract][Hide abstract] ABSTRACT: Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.
PLoS ONE 05/2013; 8(5):e64287. DOI:10.1371/journal.pone.0064287 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8(+) and CD4(+) T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8(+) and CD4(+) T cells. Unexpectedly, therapeutic blockade of PD-1-PD-L1 restored CD8(+) T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8(+) T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-γ. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8(+) T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.
Journal of Experimental Medicine 03/2013; 210(4). DOI:10.1084/jem.20121416 · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Persistent viral infections and tumors often pose a challenge to the immune system by exposing T and B cells to heightened antigenic loads and/or diverse immunoregulatory machinery. Conse quently, lymphocytes exposed to these environ ments are stricken with a state of dysfunction commonly referred to as immune exhaustion (Wherry, 2011). The term exhaustion refers to a state of functional decline that occurs when lymphocytes are chronically exposed to an anti gen. During a persistent viral infection, this is operationally defined for T cells as a progressive loss in their ability to lyse target cells and pro duce important cytokines such as IFN, TNF, and IL2 (Ahmed and Oldstone, 1988; Zajac et al., 1998; Brooks et al., 2005; Wherry et al., 2003). Exhaustion can in some instances be followed by clonal deletion, resulting in the physical removal of antiviral cells from the im mune repertoire (Moskophidis et al., 1993). It is now widely accepted that immune exhaustion contributes to the persistence of many viruses as well as tumors and is maintained by negative im mune regulators such as PD1 (Barber et al., 2006; Velu et al., 2009), IL10 (Brooks et al., 2006b; Ejrnaes et al., 2006), and CTLA4 (Kaufmann et al., 2007). Recent studies have shown that ther apeutic blockade of negative immune regulators can reverse immune exhaustion and promote clearance of both viruses and tumors (Kim and Ahmed, 2010). Immunoregulatory blockade can also be added to therapeutic vaccination regimens to improve their efficacy (Brooks et al., 2008; Ha et al., 2008). In general, immune regulators are CORRESPONDENCE Dorian B. McGavern:
[Show abstract][Hide abstract] ABSTRACT: PD-1, a receptor expressed by T-cells, B-cells and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology, but differs at the edge of the GFCC' sheet which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whilst binding is centred on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1/ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The three- to four-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the three-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T-cells with antigen presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1, and reveal that potent inhibitory signalling can be initiated by weakly interacting receptors.
[Show abstract][Hide abstract] ABSTRACT: Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
Philosophical Transactions of The Royal Society B Biological Sciences 01/2013; 368(1611):20120027. DOI:10.1098/rstb.2012.0027 · 6.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn’s disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.