Tuning DO:DM Ratios Modulates MHC Class II Immunopeptidomes

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM sensitivity, peptide abundance, binding affinity and motif, peptide length, and choice of binding register along the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.     

Promiscuous binding of invariant chain-derived CLIP peptide to distinct HLA-I molecules revealed in leukemic cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.     

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.     

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.)

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.     

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.     

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.     

Binding modes of Bcl-2 homology 3 (BH3) peptides with anti-apoptotic protein A1 and redesign of peptide inhibitors: a computational study

The interaction between protein and peptide ligand is a challenging problem in molecular biology and drug design. The binding of the Bcl-2 homology 3 (BH3) peptide to the anti-apoptotic protein A1 was revealed as a critical step in the regulation of apoptosis. These BH3 peptides hold high structural similarity, but are diverse in their regulation abilities. Based on molecular simulations and MM-P(G)BSA methods, this work presented a detailed analysis on binding mechanism of the BH3 peptides derived from PUMA and BMF. Residue-level energy decomposition showed that the core regions of BH3 peptides maintain in stable helical conformations and the four conserved hydrophobic residues together with an invariant aspartic acid contribute the major driving force for binding, whereas their two terminal segments exhibit obvious flexibility and various binding modes. Such kind of behavior was suggested as the reason for binding diversity and selectivity of BH3 peptides. As a further step, several BH3-mimetic peptides have been redesigned by computational mutation. Those new peptides showed not only stronger affinities when binding to protein A1, as well transferable binding patterns at some specific positions. A long-range coupling effect was disclosed for BH3 peptides, side-chain orientation and binding contribution of terminal residues were even affected by mutations at large sequence interval. Overall, this work reports that the binding modes of BH3 peptides are primarily dependent on its two terminal segments. The computational methods applied herein are also demonstrated to be of great assistance in the rational design of peptide inhibitors.     

Kinetics and substrate specificity of membrane-reconstituted peptide transporter DtpT of Lactococcus lactis

The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[(14)C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, omega-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT(1) and PepT(2) shows that the bacterial transporter is more restrictive in its substrate recognition.     

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.     

An accessory peptide binding site with allosteric effect on the formation of peptide-MHC-II complexes?

MHC-II molecules bind a single peptide in their groove. Here, the authors summarise evidence that a second peptide could bind transiently to MHC-II molecules outside the groove and have an allosteric effect on peptide-MHC-II complex formation. This effect could modulate, after the antigen processing, the selection of the peptide subset presented by MHC-II molecules to the helper CD4 T cells, which regulate the specific immune response.     

The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

Peptide Presentation to T Cells: Solving the Immunogenic Puzzle

The vertebrate immune system uses an impressive arsenal of mechanisms to combat harmful cellular states such as infection. One way is via cells delivering real-time snapshots of their protein content to the cell surface in the form of short peptides. Specialized immune cells (T cells) sample these peptides and assess whether they are foreign, warranting an action such as destruction of the infected cell. The delivery of peptides to the cell surface is termed antigen processing and presentation, and decades of research have provided unprecedented understanding of this process. However, predicting the capacity for a given peptide to be immunogenic—to elicit a T cell response—has remained both enigmatic and a long sought-after goal. In the era of big data, a point is being approached where the steps of antigen processing and presentation can be quantified and assessed against peptide immunogenicity in order to build predictive models. This review presents new findings in this area and contemplates challenges ahead.     

Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.     

A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain

Peptide antigens used to generate site-specific antibodies to proteins are of interest in the development of vaccines. The need to conjugate them to a carrier protein for optimal immunogenicity results in a number of problems including a possible immune response to the carrier. Here we describe a new method of synthesizing an immunogenic peptide antigen, referred to as multiple antigenic peptide (MAP), which may render the need for a carrier protein obsolete. A 14-residue sequence derived from the human T cell antigen receptor beta-chain constant region was selected, and the peptide was synthesized directly onto a branching lysine core with 8 copies of the 14-residue peptide linked to the core by the COOH-terminal amino acid. The molecular weight of this structure was 13,422 of which only 7% represents the lysine residues of the core. The octameric MAP was highly immunogenic in mice and rabbits, allowing production of polyclonal and monoclonal antibodies. The majority of these antibodies reacted with the peptide in its monomeric form as well as its octameric form. Moreover, the antibodies reacted with the intact beta-chain protein. The antigenic determinants of the peptide that were recognized by the antibodies included continuous determinants and conformational determinants. The NH2-terminal residues of the octameric MAP appeared to be most immunogenic. There were no antibodies to the central lysine core. This method of direct synthesis of a polymeric peptide provides accurate knowledge of the conformation and quantity of the peptide prior to immunization, which is usually not the case when peptides are conjugated to carriers. The method is versatile because the possibility exists to synthesize MAP with 16 or 32 peptide arms or to synthesize polymers containing two different peptides.     

Structure-guided design of peptidic ligand for human prostate specific antigen.

Prostate specific antigen (PSA) is a member of kallikrein family having serine protease-like activity and acts as a prognostic marker of prostate carcinoma. Various studies have been performed on inhibition of PSA and such targeting requires the identification of highly selective peptide inhibitors. PSA was purified from human seminal plasma by rapid and efficient methods, and binding studies for various peptides were carried out by fluorescence spectroscopy and SPR. The 'S' of PSA is predominated by hydrophobic residues, and hence many hydrophobic peptides were used to determine their binding affinity to PSA by fluorescence spectroscopy. We observed that LLFW, FFKW, and KFW binds strongly to PSA, among them LLFW showed strong binding. SPR also showed strong binding affinity of PSA toward peptides with hydrophobic and basic residues. Among the peptides used, FWYS showed dramatic increase in binding affinity (10(-10) M). The peptides analyzed for binding studies, suggests that peptide with Trp residue along with basic or hydrophobic amino acids may be useful for designing specific inhibitors for PSA. The strong affinities of designed peptides for PSA can be a valuable tool for designing therapeutic agents for prostate carcinomas.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

Structural aspects of chaperone-mediated peptide loading in the MHC-I antigen presentation pathway

Recognition of foreign and dysregulated antigens by the cellular innate and adaptive immune systems is in large part dependent on the cell surface display of peptide/MHC (pMHC) complexes. The formation of such complexes requires the generation of antigenic peptides, proper folding of MHC molecules, loading of peptides onto MHC molecules, glycosylation, and transport to the plasma membrane. This complex series of biosynthetic, biochemical, and cell biological reactions is known as “antigen processing and presentation”. Here, we summarize recent work, focused on the structural and functional characterization of the key MHC-I-dedicated chaperones, tapasin, and TAPBPR. The mechanisms reflect the ability of conformationally flexible molecules to adapt to their ligands, and are comparable to similar processes that are exploited in peptide antigen loading in the MHC-II pathway.     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94)

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.