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Biomedical subjects

C Wülfing

Publications and source records attributed to C Wülfing.

18 recordsLinked to original sources
PubMed2000

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface.

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav(-/-) mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav(+/-) mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav(-/-) mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Actins↗
PubMed2000

Thirty-six views of T-cell recognition.

While much is known about the signalling pathways within lymphocytes that are triggered during activation, much less is known about how the various cell surface molecules on T cells initiate these events. To address this, we have focused on the primary interaction that drives T-cell activation, namely the binding of a particular T-cell receptor (TCR) to peptide-MHC ligands, and find a close correlation between biological activity and off-rate; that is, the most stimulatory TCR ligands have the slowest dissociation rates. In general, TCRs from multiple histocompatibility complex (MHC) class-II-restricted T cells have half-lives of 1-11s at 25 degrees C, a much narrower range than found with antibodies and suggesting a strong selection for an optimum dissociation rate. TCR ligands with even faster dissociation rates tend to be antagonists. To observe the effects of these different ligands in their physiological setting, we made gene fusions of various molecules with green fluorescent protein (GFP), transfected them into the relevant lymphocytes, and observed their movements during T-cell recognition using multicolour video microscopy. We find that clustering of CD3zeta-GFP and CD4-GFP on the Tcell occurs concomitantly or slightly before the first rise in calcium by the T cell, and that various GFP-labelled molecules on the B-cell side cluster shortly thereafter (ICAM-1, class II MHC, CD48), apparently driven byT-cell molecules. Most of this movement towards the interface is mediated by signals through the co-stimulatory receptors, CD28 and LFA-1, and involves myosin motors and the cortical actin cytoskeleton. Thus, we have proposed that the principal mechanism by which co-stimulation enhances T-cell responsiveness is by increasing the local density of T-cell activation molecules, their ligands and their attendant signalling apparatus. In collaboration with Michael Dustin and colleagues, we have also found that the formation and stability of the TCR-peptide-MHC cluster at the centre of the interaction cap between T and B cells is highly dependent on the dissociation rate of the TCR and its ligand. Thus, we are able to link this kinetic parameter to the formation of a cell surface structure that is linked to and probably causal with respect to T-cell activation.

Animals↗
PubMed2000

Differential clustering of CD4 and CD3zeta during T cell recognition.

Whereas T helper cells recognize peptide-major histocompatibility complex (MHC) class II complexes through their T cell receptors (TCRs), CD4 binds to an antigen-independent region of the MHC. Using green fluorescent protein-tagged chimeras and three-dimensional video microscopy, we show that CD4 and TCR-associated CD3zeta cluster in the interface coincident with increases in intracellular calcium. Signaling-, costimulation-, and cytoskeleton-dependent processes then stabilize CD3zeta in a single cluster at the center of the interface, while CD4 moves to the periphery. Thus, the CD4 coreceptor may serve primarily to "boost" recognition of ligand by the TCR and may not be required once activation has been initiated.

Animals↗
PubMed1999

Visualizing lymphocyte recognition.

Studies of T cell recognition have entered new territory now that some of the basic issues of genetics, biochemistry and structure have been addressed, at least in outline form. In the present work, the focus is on a new aspect of T cell recognition that goes beyond classical biochemistry to ask, how to TCR and other cell surface molecules cooperate to initiate and control recognition?'

Animals↗
PubMed1998

A receptor/cytoskeletal movement triggered by costimulation during T cell activation.

During T cell activation, the engagement of costimulatory molecules is often crucial to the development of an effective immune response, but the mechanism by which this is achieved is not known. Here, it is shown that beads attached to the surface of a T cell translocate toward the interface shortly after the start of T cell activation. This movement appears to depend on myosin motor proteins and requires the engagement of the major costimulatory receptor pairs, B7-CD28 and ICAM-1-LFA-1. This suggests that the engagement of costimulatory receptors triggers an active accumulation of molecules at the interface of the T cell and the antigen-presenting cell, which then increases the overall amplitude and duration of T cell signaling.

Animals↗
PubMed1998

Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels.

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

B-Lymphocytes↗
PubMed1998

Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected].

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Amino Acid Sequence↗
PubMed1997

Kinetics and extent of T cell activation as measured with the calcium signal.

We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

Amino Acid Sequence↗
PubMed1997

Efficient production of heat-labile enterotoxin mutant proteins by overexpression of dsbA in a degP-deficient Escherichia coli strain.

Escherichia coli heat-labile enterotoxin (LT) mutants containing Val60-->Gly or Ser114-->Lys substitutions in the A subunit do not produce the A subunit efficiently in E. coli. These mutants accumulate mostly the B pentamer devoid of the A subunit in the periplasmic space. Here we show that overproduction of the periplasmic chaperone DsbA, which is involved in disulfide bond formation, in a strain deficient in the periplasmic protease DegP allows efficient production of the mutant LT molecules. Our results suggest that the formation of the oligomeric toxin is influenced by DsbA, which helps protein folding, and by DegP, which removes the folded intermediates that can be untoxic for the cell.

Bacterial Toxins↗
PubMed1996

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.

Amino Acid Sequence↗
PubMed1996

Altered T cell receptor ligands trigger a subset of early T cell signals.

TCR ligands are complexes of peptides and MHC proteins on the surfaces of APCs. Some of these ligands cause T cell proliferation (agonists), while others block it (antagonists). We compared the acid release, calcium flux, and proliferation response of helper T cells to a variety of ligands. We found that all agonist ligands but not most antagonist ligands trigger acid release, a general indicator of early cellular activation. Only a subset of ligands triggering acid release cause sustained calcium flux, and only a subset of these ligands cause T cell proliferation. Antagonist ligands and anti-CD4 antibodies both effectively block T cell proliferation. However, significantly greater antagonist ligand or antibody concentrations are required to block acid release and initial calcium influx. These data demonstrate a hierarchy of early T cell signaling steps and show that altered TCR ligands can initiate some steps while blocking the completion of others.

Amino Acid Sequence↗
PubMed1994

Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli. Influence of folding catalysts.

The T-cell receptor is the central recognition molecule in cellular immunity. Its extracellular domains are homologous with and thought to be structurally similar to an antibody Fab fragment. Despite the biological importance of the TCR and the ease of bacterial expression of antibody fragments, there are only few reports of TCR-fragment expression in E. coli. In order to understand the difficulties of expressing correctly folded TCR fragments in E. coli, we have characterized the expression behavior of single-chain Fv analogs of three different TCRs (scTCR). All of them can be folded into the correct conformation in the periplasm of E. coli, yet the extent of correct folding varies greatly. In order to overcome the folding problems of some of the scTCRs, we have developed a system with enhanced in vivo folding capability based on the simultaneous induction of the heat-shock response and over-expression of the E. coli disulfide isomerase DsbA at low temperature. We present a model describing the folding of the scTCRs in the periplasm of E. coli and possible points of folding assistance. The role of the periplasm as an independent folding compartment is emphasized and the existence of a general periplasmic chaperone is postulated. We have also shown that a bivalent scTCR, dimerized in vivo with helix-turn-helix modules, can be expressed in a correctly folded form.

Base Sequence↗
PubMed1994

An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif.

Initially detected as a persistent contaminant in immobilized metal affinity chromatography of recombinant proteins in Escherichia coli, a 196-amino acid protein was isolated, cloned, overexpressed, and characterized. It consists of two domains, of which the first (146 amino acids) shows some homology to the FK506-binding proteins. The second domain (50 amino acids) is extremely rich in potentially metal-binding amino acids, such as histidine, cysteine, and acidic amino acids. The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry, Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly at all.

Amino Acid Sequence↗
PubMed1994

Protein folding in the periplasm of Escherichia coli.

With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right. Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E. coli is very recent and is often speculative. This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E. coli proteins involved in this process. After an introduction to the specific physiological situation in the periplasm of E. coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation--disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones--and briefly cover the guided assembly of multi-subunit structures. Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena.

Bacterial Proteins↗
PubMed1993

A versatile and highly repressible Escherichia coli expression system based on invertible promoters: expression of a gene encoding a toxic product.

A very flexible and tightly regulatable expression system has been constructed. It uses the principle of invertible promoters [Podhajska et al., Gene 40 (1985) 163-168]. Here, we describe the construction of a plasmid that provides the integrase, which causes promoter inversion in a tightly regulated fashion, as well as modified plasmids carrying the invertible module. The way the integrase is provided on a separate plasmid closely mimicks expression of the integrase from a lambda lysogen. Thus, the flexibility of the original system is considerably extended by making it strain-independent without compromising the tight regulation. We present the expression of a single-chain T-cell receptor fragment as an example of application, in order to illustrate the properties of this expression system.

Amino Acid Sequence↗
PubMed1991

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

A protein was purified from crude extracts of the soil fungus Myrothecium verrucaria by gel filtration and hydrophobic chromatography to homogeneity; this protein catalyzed the stoichiometric hydration of the fertilizer cyanamide to urea with high substrate specificity. This cyanamide hydratase (urea hydro-lyase; EC 4.2.1.69) contained zinc and consisted of six identical subunits with Mr = 27,700. It was partially sequenced. The protein was detectable only when the fungus was grown on cyanamide as the sole nitrogen source. Genomic DNA from the fungus was cloned, and the gene encoding the enzyme was mapped with an oligonucleotide probe derived from the amino acid sequence within a 25,800-base-pair DNA region. The subunit of the enzyme is encoded by a 795-base-pair DNA sequence containing a 63-base-pair intron. A cDNA clone containing the intronless gene with an open reading frame encoding a sequence of 244 amino acids expressed the enzyme in active form in Escherichia coli with excellent yield.

Amino Acid Sequence↗