Structural requirements and biological significance of interactions between peptides and the major histocompatibility complex

Previous studies indicate that T cells recognize a complex between the major histocompatibility complex (MHC) restriction-element and peptide-antigen fragments. Two aspects of this complex formation are considered in this paper: (1) what is the nature of the specificity of the interactions that allows a few MHC molecules to serve as restriction elements for a large universe of antigens; and (2) what is the relative contribution of determinant selection (i.e. antigen-MHC complex formation) and T-cell repertoire in determining the capacity of an individual to respond to an antigen? By analysing single amino acid substitution analogues of a peptide antigen (Ova 325-335) as well as by analysing the structural similarities between unrelated peptides capable of binding to the same MHC molecule, we have been able to document the very permissive nature of the antigen-MHC interaction. Despite this permissiveness of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity. With respect to the question of the relative role of 'determinant selection' and 'holes in the T-cell repertoire' in determining immune responsiveness, we present data that suggest both mechanisms operate in concert with one another. Thus only about 30% of a collection of peptides that in sum represent the sequence of a protein molecule were found to bind to Ia. Although immunogenicity was restricted to those peptides that were capable of binding to Ia (i.e. determinant selection was operative), we found that about 40% of Ia-binding peptides were not immunogenic (i.e. there were also 'holes in the T-cell repertoire').     

Iterative stepwise discriminant analysis: a meta-algorithm for detecting quantitative sequence motifs.

An algorithm is presented for detecting a quantitative pattern in peptide fragments that bind class II major histocompatibility complex (MHC) molecules. It is referred to as a meta-algorithm because it requires successive applications of Stepwise Discriminate Analysis (SDA). On every iteration the best subsequence candidates are selected from sequences known to bind class II MHC molecules. When SDA compares probable binding subsequences with subsequences known not to bind class II MHC molecules, a quantitative model emerges that is capable of classifying subsequences as binding or non-binding. In an iterative manner, the resultant model is utilized as a criterion for selecting probable binding subsequence candidates. The procedure is repeated until models converge. In the illustrated examples, the final models correctly classify over 95% of the peptides in a database of peptides whose binding affinity for HLA-DR1 is known. The final model can then be used to predict the binding affinity of peptides that have not yet been laboratory tested.     

Selection of RNA-binding peptides using mRNA-peptide fusions

We have been working to apply in vitro selection to isolate novel RNA-binding peptides. To do this, we use mRNA-protein fusions, peptides covalently attached to their own mRNA. Here, we report selection protocols developed using the arginine-rich domain of bacteriophage lambda-N protein and its binding target, the boxB RNA. Systematic investigation of possible paths for a selection round has allowed us to design a reliable and efficient protocol to enrich RNA-binding peptides from nonfunctional members of a complex mixture. The protocols we have developed should greatly facilitate the isolation of new molecules using the fusion system.     

Receptor mediated uptake of peptides that bind the human transferrin receptor.

A biopanning process designed to find peptide epitopes specific for cell surface receptors has been used in this study to select seven- and 12-amino-acid peptides capable of binding to and internalizing with the human transferrin receptor (hTfR). Through sequential rounds of negative and positive selection, two peptide sequences were identified that specifically bind to the hTfR. Phage containing the sequences HAIYPRH or THRPPMWSPVWP were inhibited from binding the hTfR in a dose-dependent fashion when peptides of the same sequence were present in a competition assay. Interestingly, transferrin did not compete with either of these sequences for receptor binding, suggesting that these peptides bind a site on the hTfR distinct from the transferrin binding site. When either of these sequences was expressed as a fusion to green fluorescent protein (GFP), the recombinant GFP molecule was internalized in cells expressing the hTfR. These studies suggest that the two peptides can be used to target other proteins into the endosomal pathway. Further, they provide a strategy for identifying peptides that bind to other cell surface receptors that can be used for both diagnostic and therapeutic purposes.     

Corruption of phage display libraries by target-unrelated clones: Diagnosis and countermeasures

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.     

噬菌体展示技术及其在肿瘤研究中的应用

噬菌体表面展示技术是一项特异性多肽或蛋白的筛选技术,它将随机序列的多肽或蛋白片段与噬菌体衣壳蛋白融合表达而呈现于病毒表面,被展示的多肽能保持相对独立的空间结构,使其能够与配体作用而达到模仿性筛选特异性分子表位,从而提供了高通量高效率的筛选系统。近年来噬菌体展示技术已广泛应用于肿瘤抗原抗体库的建立、单克隆抗体制备、多肽筛选、疫苗研制、肿瘤相关抗原筛选和抗原表位研究、药物设计、癌症检测和诊断、基因治疗及细胞信号转导研究等。就近年来噬菌体展示技术在肿瘤相关研究中的运用作以综述。     

Encoded self-assembling chemical libraries

The isolation of molecules capable of high-affinity and specific binding to biological targets is a central problem in chemistry, biology and pharmaceutical sciences. Here we describe the use of encoded self-assembling chemical (ESAC) libraries for the facile identification of molecules that bind macromolecular targets. ESAC technology uses libraries of organic molecules linked to individual oligonucleotides that mediate the self-assembly of the library and provide a code associated with each organic molecule. After panning ESAC libraries on the biomolecular target of interest, the 'binding code' of the selected compounds can be 'decoded' by a number of experimental techniques (e.g., hybridization on oligonucleotide microarrays). The potential of this technology was demonstrated by the affinity maturation (>40-fold) of binding molecules to human serum albumin and bovine carbonic anhydrase, leading to binders with dissociation constants in the nanomolar range.     

Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.

MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.     

Recombinant antibodies for the depletion of abundant proteins from human serum

The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.     

NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

Background  

The major histocompatibility complex (MHC) molecule plays a central role in controlling the adaptive immune response to infections. MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells, whereas MHC class II molecules stimulate cellular and humoral immunity through presentation of extracellularly derived peptides to helper T cells. Identification of which peptides will bind a given MHC molecule is thus of great importance for the understanding of host-pathogen interactions, and large efforts have been placed in developing algorithms capable of predicting this binding event.     

食用菌SSR序列开发策略研究进展

SSR序列开发策略主要有3大类:传统的基因文库筛选法、GenBank筛选法和富集文库筛选法。文中在对传统的基因文库筛选法、GenBank筛选法的优缺点及在食用菌SSR分子标记的开发应用进行综述的基础上,重点综述了富集文库筛选法,即基于RAPD技术的SSR开发策略、SSR兼并引物法、序列标签法构建SSR富集文库、vectorette PCR染色体步移法分离SSR位点、Suppression PCR染色体步移法分离SSR位点、引物延伸法构建SSR富集文库以及SSR杂交捕获法构建SSR富集文库的具体设计原理和步骤,并提出食用菌中SSR分子标识的开发现状和展望。     

胰凝乳蛋白酶短肽抑制剂的筛选和活性分析

报道了一种从噬菌体肽库中筛选胰凝乳蛋白酶短肽抑制剂的新方法．在通常的亲和富集筛选的基础上,利用胰凝乳蛋白酶自身的水解活力切割掉结合的底物噬菌体,再经抑制活力分析得到抑制性噬菌体克隆．这样筛得的噬菌体克隆具有明显的胰凝乳蛋白酶结合活力和抑制活力,ＤＮＡ序列分析发现其保守序列为（Ｓ／Ｔ）ＲＶＰＲ（Ｒ／Ｈ）．按此序列化学合成的短肽Ａｃ－ＡＳＲＶＰＲＲＧ－ＮＨ２、Ａｃ－ＡＳＲＶＰＲＨＧ－ＮＨ２同样表现出对胰凝乳蛋白酶的抑制作用．该方法为蛋白酶短肽抑制剂的筛选提供了一条有效途径     

Diversity visualization by endonuclease: a rapid assay to monitor diverse nucleotide libraries

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a single-strand specific nuclease. The assay involves only a limited number of steps and can be performed in less than 4 h, including the initial PCR. After PCR, the homoduplex double-stranded DNA (dsDNA) is denatured and reannealed under stringent conditions. During the reannealing process, incubation with S1 nuclease removes single-stranded loops of formed heteroduplexes and the resulting digest is visualized on agarose gel. The sequence diversity is inversely proportional to the band intensities of S1 nuclease surviving dsDNA molecules of expected size. As an example, we employed DiVE to monitor the diversity of panning rounds from a single-framework, semisynthetic single-chain antibody fragment (scFv) phage display library. The results are in good agreement with the observed decrease in diversity in phage display panning rounds toward the selection of monoclonal scFv. We conclude that the DiVE assay allows rapid and cost-effective monitoring of diversities of various nucleotide libraries and proves to be particularly suitable for scaffold-based randomized libraries.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.     

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.     

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D -amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.     

Template selection during manipulation of complex mixtures by PCR

PCR is ubiquitous in molecular biology. It is used to amplify single sequences from large genomes, or populations of sequences from complex mixtures such as cDNA libraries in mammalian cells. These cDNA libraries are often employed in subsequent labor-intensive experiments such as genetic screens, the outcome of which depends on library quality. The use of PCR to amplify diverse sequence populations raises important technical issues. One question is whether or not PCR is capable of maintaining population diversity, specifically with respect to template selection in the first rounds of the amplification process (i.e., the possibility that rare sequences in a complex mixture are lost because of amplification failure at the outset of the PCR). Here, we analyze the properties of PCR in the context of template selection in complex mixtures and show that it is an excellent method for preserving diversity.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Accurate Prediction of Peptide Binding Sites on Protein Surfaces

Many important protein–protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled) but where no structure of the protein–peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein–peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.