CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms

Recently, we reported that soluble CD83 has a strong immunosuppressive activity in vitro as well as in vivo. Sequence alignment of CD83 between different species revealed the presence of five cysteines in the extracellular Ig-domain of the protein. This opens up the possibility that four cysteines are involved in the formation of two intramolecular disulfide bonds and a possible involvement of the remaining fifth cysteine in the formation of an intermolecular covalent disulfide bond, leading to the dimerization of the extracellular protein domains. Using recombinant mutational analyses, where the fifth cytosine at amino acid position 129 was mutated to a serine, we could prove that the fifth cysteine residue was indeed necessary for the dimerization. Functional analyses revealed that the mutant protein inhibited almost completely the upregulation of CD83-expression during DC maturation. Furthermore, the functional activity of the mutant protein was investigated using MLR assays and we could show that the mutant soluble CD83 protein inhibited DC-mediated allogeneic T-cell stimulation in vitro.     

Isolation, folding and structural investigations of the amino acid transporter OEP16

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni–NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.     

Heterodimeric CD3epsilongamma extracellular domain fragments: production, purification and structural analysis

The CD3 polypeptides (epsilon, gamma, and delta) are non-covalently associated signaling subunits of the T cell receptor which form non-disulfide linked epsilongamma and epsilondelta heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3epsilon subunit N-terminal Ig-like extracellular domain alone or as a single chain construct with that of CD3gamma. The latter links the CD3gamma segment to the C terminus of the CD3epsilon segment via a 26 amino acid peptide (scCD3epsilongamma26). Although CD3epsilon could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3epsilon was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by (15)N-(1)H two-dimensional NMR analysis. In contrast, scCD3epsilongamma26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3epsilon and CD3gamma is critical since scCD3epsilongamma16 containing a 16 residue connector failed to generate a stable heterodimer. Collectively, the results demonstrate that: (i) soluble heterodimeric fragments of CD3 can be produced; (ii) cotranslation of CD3 chains insures proper folding even in the absence of the conserved ectodomain stalk region (CxxCxE); and (iii) CD3epsilon has a more stable tertiary protein fold than CD3gamma.     

Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria

Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.     

Stable, soluble T-cell receptor molecules for crystallization and therapeutics

Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.     

Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.     

Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles

Protein antigen (Ag)-based immunotherapies have the advantage to induce T cells with a potentially broad repertoire of specificities. However, soluble protein Ag is generally poorly cross-presented in MHC class I molecules and not efficient in inducing robust cytotoxic CD8⁺ T cell responses. In the present study, we have applied poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which strongly improve protein Ag presentation by dendritic cells (DC) in the absence of additional Toll-like receptor ligands or targeting devices. Protein Ag-loaded DC were used as antigen presenting cells to stimulate T cells in vitro and subsequently analyzed in vivo for their anti-tumor effect via adoptive transfer, a treatment strategy widely studied in clinical trials as a therapy against various malignancies. In a direct comparison with soluble protein Ag, we show that DC presentation of protein encapsulated in plain PLGA-NP results in efficient activation of CD4⁺ and CD8⁺ T cells as reflected by high numbers of activated CD69⁺ and CD25⁺, interferon (IFN)-γ and interleukin (IL)-2-producing T cells. Adoptive transfer of PLGA-NP-activated CD8⁺ T cells in tumor-bearing mice displayed good in vivo expansion capacity, potent Ag-specific cytotoxicity and IFN-γ cytokine production, resulting in curing mice with established tumors. We conclude that delivery of protein Ag through encapsulation in plain PLGA-NP is a very efficient and simple procedure to stimulate potent anti-tumor T cells.     

Conformational flexibility of alpha-lactalbumin related to its membrane binding capacity

Different folding states of the small, globular milk protein bovine alpha-lactalbumin (BLA) induced by the anionic surfactant sodium dodecylsulphate (SDS) have been examined by fluorescence spectroscopy, CD and NMR. The solution structure of the protein in the absence of SDS was also determined, indicating fluidity even under native conditions. BLA is partly denatured to a molten globule (MG)-like state by micromolar concentrations of SDS, and the transitions from native to MG-like state are dependent on pH, the protein being more sensitive to the surfactant at pH 6.5. As indicated by measurements of the intrinsic emission fluorescence, the tertiary structure disappears at lower concentrations of SDS than most of the secondary structure, as estimated from CD data. The MG-like state induced by low concentrations of SDS is not observable by NMR, and is probably fluctuating and/or aggregating. At higher concentrations of SDS above the critic concentration of micelles, an NMR-observable state reappears. This micelle-associated conformer was partially assigned, and found to bear strong resemblance to the acid-tri-fluoroethanol state, retaining weakened versions of the A and C helix of native BLA. We discuss the results in terms of the inherent flexibility of the protein, and its ability to form multiple folding states and to bind to membranes. Also, we propose that proteins with stable MG-like conformers can have these states stabilized by low levels of compounds with surfactant properties in vivo.     

Quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-gamma production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c(+) DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 microm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-gamma production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.     

Folding and Stability of Chimeric Immunofusion VL-Barstar

A chimeric protein VL-barstar that comprises the VL domain of anti-human ferritin monoclonal antibody F11 and barstar, the naturally occurring inhibitor of bacterial RNase barnase, has been constructed for study of structure-function characteristics of chimeric immunoglobulin fused proteins. Such chimeric constructs may be potentially employed for development of bivalent/bispecific antibodies on the basis of the high affinity interaction between barstar and barnase (the association constant is about 10(14) M(-1)). We have developed a protocol for VL-barstar expression in E. coli and purification and refolding from inclusion bodies that yields a homogeneous and soluble form of this protein. Differential scanning calorimetry in combination with fluorescence and CD spectroscopy revealed that the VL-barstar formed well-resolved ordered secondary and compact tertiary structures. However, partial loss of tertiary interactions resulted in low stability of the recombinant protein and the lack of functional activity of the two constituent modules. These conformational features suggest that the protein might be referred to the class of native molten globules, which comprises partially unfolded conformations stabilized under physiological conditions. Since individually expressed VL domain and barstar retain completely folded conformation and stable spatial structure, the incomplete folding of the chimeric protein may be attributed to interaction between heterologous domains, which appears at the folding stage preceding formation of a system of tertiary interactions in both structural modules. The results provide evidence for non-native interactions between heterologous modules that may occur in chimeric proteins composed of taxonomically distinct fusion partners.     

Enhanced immune responses in transgenic mice expressing a truncated form of the lymphocyte semaphorin CD100

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5(+) B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.     

The use of osmolytes to facilitate protein NMR spectroscopy

Summary A method of stabilizing folded proteins is described, which allows NMR studies under conditions where a protein would normally be unfolded. This enables stable proteins to be examined at elevated temperatures, or spectra recorded on samples that are insufficiently stable under normal conditions. Up to two molar perdeuterated glycine, a potent osmolyte, can be added to aqueous protein NMR samples without altering the folded three-dimensional structure or function of the protein. However, the stability of the folded form is dramatically increased. This is illustrated for the protein lysozyme at high temperature (348 K) where the structural integrity is destroyed in standard aqueous solution, but is retained in the osmolyte solution. We hope that the technique will be of value to those studying by NMR the structural biology of protein fragments and mutants, which are often of reduced stability compared with the original proteins.To whom correspondence should be addressed.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor interaction

Tumor necrosis factor (TNF) exists both as a membrane-integrated type II precursor protein and a soluble cytokine that have different bioactivities on TNFR2 (CD120b) but not on TNFR1 (CD120a). To identify the molecular basis of this disparity, we have investigated receptor chimeras comprising the cytoplasmic part of Fas (CD95) and the extracellular domains of the two TNF receptors. The membrane form of TNF, but not its soluble form, was capable of inducing apoptosis as well as activation of c-Jun N-terminal kinase and NF-kappaB via the TNFR2-derived chimera. In contrast, the TNFR1-Fas chimera displayed strong responsiveness to both TNF forms. This pattern of responsiveness is identical to that of wild type TNF receptors, demonstrating that the underlying mechanisms are independent of the particular type of the intracellular signaling machinery and rather are controlled upstream of the intracellular domain. We further demonstrate that the signaling strength induced by a given ligand/receptor interaction is regulated at the level of adaptor protein recruitment, as shown for FADD, caspase-8, and TRAF2. Since both incidents, strong signaling and robust adapter protein recruitment, are paralleled by a high stability of individual ligand-receptor complexes, we propose that half-lives of individual ligand-receptor complexes control signaling at the level of adaptor protein recruitment.     

Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.     

In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina

We have proposed that the [Het-s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues. We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein. The HET-s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type [Het-s] strains. The antagonistic HET-S form is soluble even at high expression level. A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het-s] element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein.     

Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.     

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle

Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase M_B and water soluble form M_A.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms M_A and M_B share the same protein as shown in immunodiffusion test with the anti-M_A sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form M_B is rapidly inactivated whereas form M_A is quite stable under the same conditions. Both forms have different K_m for phosphoenolpyruvate and ADP and V_max as well.Digestion of pyruvate kinase M_B with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences.