Enhancing multiple disulfide bonded protein folding in a cell-free system

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.     

Comparative molecular dynamics analysis of tapasin-dependent and -independent MHC class I alleles

MHC class I molecules load antigenic peptides in the endoplasmic reticulum and present them at the cell surface. Efficiency of peptide loading depends on the class I allele and can involve interaction with tapasin and other proteins of the loading complex. Allele HLA-B*4402 (Asp at position 116) depends on tapasin for efficient peptide loading, whereas HLA-B*4405 (identical to B*4402 except for Tyr116) can efficiently load peptides in the absence of tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Comparative unrestrained molecular dynamics simulations on the alpha(1)/alpha(2) peptide binding domains performed in the presence of bound peptides resulted in structures in close agreement with experiments for both alleles. In the absence of peptides, allele-specific conformational changes occurred in the first segment of the alpha(2)-helix that flanks the peptide C-terminal binding region (F-pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B*4402, a shift toward an altered F-pocket structure deviating significantly from the bound form was observed. Subsequent free energy simulations on induced F-pocket opening in B*4402 confirmed a conformation that deviated significantly from the bound structure. For B*4405, a free energy minimum close to the bound structure was found. The simulations suggest that B*4405 has a greater tendency to adopt a peptide receptive conformation in the absence of peptide, allowing tapasin-independent peptide loading. A possible role of tapasin could be the stabilization of a peptide-receptive class I conformation for HLA-B*4402 and other tapasin-dependent alleles.     

Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623-644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361-369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.     

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.     

Specificity of amyloid precursor-like protein 2 interactions with MHC class I molecules

The ubiquitously expressed amyloid precursor-like protein 2 (APLP2) has been previously found to regulate cell surface expression of the major histocompatibility complex (MHC) class I molecule K(d) and bind strongly to K(d). In the study reported here, we demonstrated that APLP2 binds, in varied degrees, to several other mouse MHC class I allotypes and that the ability of APLP2 to affect cell surface expression of an MHC class I molecule is not limited to K(d). L(d), like K(d), was found associated with APLP2 in the Golgi, but K(d) was also associated with APLP2 within intracellular vesicular structures. We also investigated the effect of beta(2)m on APLP2/MHC interaction and found that human beta(2)m transfection increased the association of APLP2 with mouse MHC class I molecules, likely by affecting H2 class I heavy chain conformation. APLP2 was demonstrated to bind specifically to the conformation of L(d) having folded outer domains, consistent with our previous results with K(d) and indicating APLP2 interacts with the alpha1alpha2 region on each of these H2 class I molecules. Furthermore, we observed that binding to APLP2 involved the MHC alpha3/transmembrane/cytoplasmic region, suggesting that conserved as well as polymorphic regions of the H2 class I molecule may participate in interaction with APLP2. In summary, we demonstrated that APLP2's binding, co-localization pattern, and functional impact vary among H2 class I molecules and that APLP2/MHC association is influenced by multiple domains of the MHC class I heavy chain and by beta(2)m's effects on the conformation of the heavy chain.     

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

Partial purification and characterization of a novel human factor that augments the expression of class I MHC antigens on tumour cells

A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC- AF) has been partially purified by Sephadex G- 100 column chromatography, preparative isoelectric focusing and HPLC with ion- exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6.0. Interferon (IFN)-α, \, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC- AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-\ and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2.0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.     

A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase.

Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 1/256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.     

Crystal structure of a non-canonical high affinity peptide complexed with MHC class I: a novel use of alternative anchors

The crystal structure of a non-standard peptide, YEA9, in complex with H-2Kb, at 1.5 A resolution demonstrates how YEA9 peptide can bind with surprisingly high affinity through insertion of alternative, long, non-canonical anchors into the B and E pockets. The use of "alternative pockets" represents a new mode of high affinity peptide binding, that should be considered when predicting peptide epitopes for MHC class I. These novel interactions encountered in this non-canonical high affinity peptide-MHC complex should help predict additional binding peptides from primary protein sequences and aid in the design of alternative approaches for peptide-based vaccines.     

Potential roles of protein oxidation and the immunoproteasome in MHC class I antigen presentation: the 'PrOxI' hypothesis

The major histocompatibility complex (MHC) class I (MHC-I) antigen presentation system is responsible for the cell-surface presentation of self-proteins and intracellular viral proteins. This pathway is important in screening between self, and non-self or infected cells. In this pathway, proteins are partially degraded to peptides in the cytosol and targeted to the cell surface bound to an MHC-I receptor protein. At the cell surface, T cells bypass cells displaying self-peptides but destroy others displaying foreign antigens. Cells contain several isoforms of the proteasome, but it is thought that the immunoproteasome is the major form involved in generating peptides for the MHC-I pathway. How all intracellular proteins are targeted for MHC-I processing is unclear. Oxidative stress is experienced by all cells, and all proteins are exposed to oxidation. We propose that oxidative modification makes proteins susceptible to degradation by the immunoproteasome. This could be called the protein oxidation and immunoproteasome or 'PrOxI' hypothesis of MHC-I antigen processing. Protein oxidation may, thus, be a universal mechanism for peptide generation and presentation in the MHC-I pathway.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

Design of enhanced agonists through the use of a new virtual screening method: application to peptides that bind class I major histocompatibility complex (MHC) molecules

A new screening procedure is described that uses docking calculations to design enhanced agonist peptides that bind to major histocompatibility complex (MHC) class I receptors. The screening process proceeds via single mutations of one amino acid at the positions that directly interact with the MHC receptor. The energetic and structural effects of these mutations have been studied using fragments of the original ligand that vary in length. The results of these docking studies indicate that the mutant affinity ranking of long peptides can be practically reproduced with a screening approach performed using fragments of six residues. Fragments of four and five residues could mimic, in some cases, the structural arrangement of the side chains of the full-length peptide. We have compared the structural and energetic results of the docking calculations with experimental data using three unrelated ligand peptides that differ greatly in their affinity for the MHC complex. Analysis of the affinity of the fragments led to the identification of three important parameters in the construction of fragments that mimic the structural and energetic properties of the full-length ligand: the length of the fragment; its intermolecular energy; and the number and localization, internal or terminal, of the anchor residues. The results of this new peptide-design methodology have been applied to suggest new peptides derived from the MUC1-8 peptide that could be used as murine vaccines that trigger the immune response through the MHC class I protein H-2K(b).