RESUMO
The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the α2-1-helix of MHC-I, 'loosening' the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11-20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.
Assuntos
Alelos , Apresentação de Antígeno/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regulação Alostérica , Biocatálise , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Antígenos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Imunoglobulinas/ultraestrutura , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/ultraestrutura , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Biblioteca de Peptídeos , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.
Assuntos
Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Linhagem Celular , Membrana Celular/imunologia , Cromatografia Líquida/métodos , Antígenos HLA/imunologia , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/química , Proteínas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Understanding the most appropriate workflow for biochemical human leukocyte antigen (HLA)-associated peptide enrichment prior to ligand sequencing is essential to achieve optimal sensitivity in immunopeptidomics experiments. The use of different detergents for HLA solubilization as well as complementary workflows to separate HLA-bound peptides from HLA protein complex components after their immunoprecipitation including HPLC, C18 cartridge, and 5 kDa filter are described. It is observed that all solubilization approaches tested led to similar peptide ligand identification rates; however, a higher number of peptides are identified in samples lysed with CHAPS compared with other methods. The HPLC method is superior in terms of HLA-I peptide recovery compared with 5 kDa filter and C18 cartridge peptide purification methods. Most importantly, it is observed that both the choice of detergent and peptide purification strategy creates a significant bias for the identified peptide sequences, and that allele-specific peptide repertoires are affected depending on the workflow of choice. The results highlight the importance of employing a suitable strategy for HLA peptide enrichment and that the obtained peptide repertoires do not necessarily reflect the true distributions of peptide sequences in the sample.
Assuntos
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Detergentes/química , Antígenos HLA/imunologia , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Proteoma/imunologiaRESUMO
In lieu of peptides, the monomorphic MHC-I-like molecule MR1 presents small molecule antigens to stimulate a subset of αß T cells known as mucosal-associated (semi-) invariant T (MAIT) cells or, more broadly, MR1-restricted (MR1T) cells. The MR1 ligands identified to date are limited to derivatives and intermediates of the riboflavin and folate biosynthesis pathways and their presentation is therefore thought to be an indicator of infection by microbial species that can synthesize riboflavin. MAIT cells have, in recent years, been studied and isolated using a tetrameric reagent of recombinant MR1 loaded with the canonical ligand 5-OP-RU due to its potency toward MAIT clones. However, new evidence has shown that the repertoire of MR1 ligands is much more diverse than previously appreciated and, consistent with this, that the 5-OP-RU tetramer does not bind all MR1T cells. To study MR1-restricted T cell clones in the context of unique bacterial infection, we have generated a tetramer of MR1 loaded with diverse microbial antigens. The production of this reagent is detailed in this chapter.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/imunologia , Corpos de Inclusão , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/imunologia , Multimerização Proteica , Proteínas Recombinantes , Linhagem Celular , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Corpos de Inclusão/metabolismo , Ligantes , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/isolamento & purificação , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Antígenos de Histocompatibilidade Classe I/genética , Interleucina-23/genética , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Análise Fatorial , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Corpos de Inclusão/química , Interleucina-23/química , Interleucina-23/isolamento & purificação , Isopropiltiogalactosídeo/farmacologia , Análise de Componente Principal , Redobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , SolubilidadeRESUMO
The large-scale and in-depth identification of MHC class I- and II-presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as for developing innovative immunotherapeutic approaches against cancer and other diseases. In this chapter we briefly review the existing strategies for the isolation of MHC-restricted peptides and provide a detailed protocol for the immunoaffinity purification of MHC class I- and II-presented peptides from primary tissues or cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Biologia Molecular/métodos , Cromatografia de Afinidade , Cromatografia Líquida , Humanos , Ligantes , Peptídeos/metabolismo , Espectrometria de Massas em TandemRESUMO
The major histocompatibility complex (MHC) is a term for all gene groups of a major histocompatibility antigen. It binds to peptide chains derived from pathogens and displays pathogens on the cell surface to facilitate T-cell recognition and perform a series of immune functions. MHC molecules are critical in transplantation, autoimmunity, infection, and tumor immunotherapy. Combining machine learning algorithms and making full use of bioinformatics analysis technology, more accurate recognition of MHC is an important task. The paper proposed a new MHC recognition method compared with traditional biological methods and used the built classifier to classify and identify MHC I and MHC II. The classifier used the SVMProt 188D, bag-of-ngrams (BonG), and information theory (IT) mixed feature representation methods and used the extreme learning machine (ELM), which selects lin-kernel as the activation function and used 10-fold cross-validation and the independent test set validation to verify the accuracy of the constructed classifier and simultaneously identify the MHC and identify the MHC I and MHC II, respectively. Through the 10-fold cross-validation, the proposed algorithm obtained 91.66% accuracy when identifying MHC and 94.442% accuracy when identifying MHC categories. Furthermore, an online identification Web site named ELM-MHC was constructed with the following URL: http://server.malab.cn/ELM-MHC/ .
Assuntos
Biologia Computacional , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Aprendizado de Máquina , Algoritmos , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/classificação , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Internet , SoftwareRESUMO
Comprehensive knowledge of the HLA class I and class II peptides presented to T cells is crucial for designing innovative therapeutics against cancer and other diseases. So far, methodologies for recovery of HLA class I and II peptides for subsequent mass spectrometry-based analysis have been a major limitation. In this chapter we describe a detailed protocol for a high-throughput, reproducible, and sensitive immunoaffinity-purification of HLA-I and HLA-II peptides from up to 96 samples in a plate format, suitable for tissue samples and cell lines. Our methodology reduces sample handling, has a competitive peptide yield, and can be completed within 5 h. This simplified pipeline is applicable for basic and clinical applications.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Neoplasias/imunologia , Peptídeos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Humanos , Hibridomas , Neoplasias/patologia , Peptídeos/imunologia , Espectrometria de Massas em Tandem/instrumentação , Fatores de TempoRESUMO
Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry.
Assuntos
Antígenos de Neoplasias/isolamento & purificação , Exoma/imunologia , Neoplasias/imunologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Exoma/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Humanos , Hibridomas , Imunoprecipitação/instrumentação , Imunoprecipitação/métodos , Neoplasias/patologia , Proteômica/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
BACKGROUND: Currently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified. RESULTS: A complex of SLA-I constituted by an SLA-2*HB01 molecule with swine ß2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64-72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å. CONCLUSION: This research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.
Assuntos
Epitopos , Vírus da Febre Aftosa/genética , Antígenos de Histocompatibilidade Classe I , Modelos Moleculares , Difração de Raios X , Animais , Cristalização , Epitopos/química , Epitopos/genética , Epitopos/isolamento & purificação , Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/metabolismo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Sorogrupo , Suínos , Linfócitos T Citotóxicos/metabolismoRESUMO
Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, a comparison of these approaches for the isolation of two different B-cell lymphoblast cell models is presented, and it is reported on the recovery, reproducibility, scalability, and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4-fold increase in MIPs compared to the MAE. The comprehensiveness of these immunopeptidome analyses is extended using personalized genomic database of B-cell lymphoblasts, and it is discovered that 0.4% of their respective MIP repertoire harbored nonsynonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs).
Assuntos
Ácidos/química , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Imunoprecipitação/métodos , Fragmentos de Peptídeos/isolamento & purificação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
A new MICA allelic variant, MICA*012:05, has been identified in a Chinese Mongolian population. Following polymerase chain reaction-sequence-based typing (PCR-SBT), this new allele was further confirmed by cloning and sequencing. MICA*012:05 was linked to an HLA-A*24-C*01-B*55:02-DRB1*09 haplotype. MICA*012:05 differs from MICA*012:01 by a single synonymous C to T substitution at nucleotide position 269 in exon 3.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Alelos , Povo Asiático , China , Éxons/genética , Haplótipos/genética , Haplótipos/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , HumanosRESUMO
Major histocompatibility complex (MHC) class I-related chain A/B (MICA/B) is a type of stress-induced molecule that plays an important role in tumor surveillance. MICA/B shares a similar structure with MHC class I molecules, but MICA/B contains a closed cleft, not an open one, in its N-terminal alpha1 domain. The alpha 1 domain was believed to have no roles in antigen presentation, because the closed cleft provides limited space for binding with known molecules, and the cleft of MICA/B have been reported no known functions. To study the possible function of the cleft located in human MICA/B's alpha 1 domain, we attempted to express the human MICB-α1 (hMICB-α1) domain allele protein, which is approximately 20.5 kDa, by utilizing an Escherichia coli (E. coli) secretory pathway. Protein expression was accomplished through the phosphate-limited inducible promoter. After purification using ammonium sulfate precipitation, phenyl hydrophobic Sepharose, SP Sepharose and HisTrap affinity Sepharose, recombinant human MICB-α1 (rhMICB-α1) was obtained with 94.3% purity. The binding capacity of rhMICB-α1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-α1 can be prepared through the E. coli secretory pathway. Purified rhMICB-α1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-α1 protein, which can served as the basis for further studies of the possible function of the MICB cleft.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Sequência de Aminoácidos , Escherichia coli/genética , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Plasmídeos/genética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Corpos de Inclusão/química , Subfamília K de Receptores Semelhantes a Lectina de Células NK/química , Redobramento de Proteína , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Corpos de Inclusão/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/isolamento & purificação , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , SolubilidadeRESUMO
Most current methods for identifying peptides that are bound to a distinct MHC-I product in a given cell sample utilize detergent solubilization of membrane proteins followed by immunoaffinity purification. Since detergent traces and cell debris hamper subsequent peptide analysis, exceedingly large cell samples are often required. To avoid the use of detergents, truncated MHC-I heavy chains have recently been expressed by stable DNA transfection or retroviral transduction, resulting in the secretion of soluble MHC-I complexes to the growth medium. The electroporation of in vitro-transcribed mRNA achieves remarkable efficacy and uniformity of gene expression in numerous cell types, exhibiting exceedingly fast kinetics. We reasoned that mRNA transfection offers a simple, fast and widely applicable alternative to current gene delivery protocols for expressing secreted MHC-I products in cells of interest. To test this assumption we used mRNA to express soluble derivatives of HLA-A2 in the human AF10 B cell myeloma and 624mel melanoma and H-2K(d) in the mouse SP2/0 B cell myeloma. The level of MHC-I complexes secreted by these cells peaked within less than 24h post-transfection and they could be affinity-purified directly from the culture medium in considerably greater yields when compared to nonionic detergent lysates on a cell-to-cell basis. Mass-spectrometry analysis of eluted peptides revealed larger pools in the secreted material than in lysates with substantial overlap in composition. Our results introduce mRNA transfection as a powerful tool for determining the cell's MHC-I peptidome, which can be potentially applied to a broad range of cell types.
Assuntos
Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , RNA Mensageiro/genética , Proteínas Recombinantes , Animais , Linhagem Celular , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Camundongos , TransfecçãoRESUMO
The chicken major histocompatibility complex (MHC) class I molecules named BF are strongly associated with Marek's disease (MD). A single structure, that of chicken BF2*2101 from the B21 haplotype, which might provide resistance to MD, has been determined. However, little is known about other structures apart from BF2*2101. In order to provide further structures of chicken MHC class I molecules, BF2*1501 and chicken ß(2)-microglobulin complexed with a nonapeptide (MDV-MEQ(RRR9)) derived from Marek's disease virus MEQ protein (MDV EcoRI Q fragment, residues 72-80) were assembled and crystallized. Diffraction data from the crystal were collected to 2.6 Å resolution; the crystal belonged to space group P3(1)21, with unit-cell parameters a = 125.1, b = 125.1, c = 80.9 Å and two molecules in the asymmetric unit. The Matthews coefficient V(M) was 2.08 Å(3) Da(-1), with a calculated solvent content of 40.78%. These data will be helpful in obtaining insight into the structural basis of the involvement of BF2*1501 in MD.
Assuntos
Galinhas/imunologia , Antígenos de Histocompatibilidade Classe I/química , Animais , Cristalização , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Redobramento de ProteínaRESUMO
The MICA Project of the 16th International HLA and Immunogenetics Workshop was planned to improve the detection of specific antibodies against MICA antigens that develop in organ transplant recipients. It was organized as a serum exchange with 18 laboratories located in seven countries as participants. Each laboratory used the MICA screening reagents available to them. It was found that there were four different kits of Luminex beads conjugated with MICA antigens that could be used in these experiments. They were, kit BL, produced by Rainer Blasczyk in Hannover, Germany, containing 12 MICA antigens, the kit from Gen-Probe (GP), which included 28 MICA antigens, the beads from One Lambda (OL), consisting of ten antigens, and the beads produced in the laboratory of the organizers (ZS) consisting of 11 MICA antigens. The sera were all from transplant recipients and represented a spectrum of MICA-specific antibodies that are commonly found. All laboratories accurately could recognize which sera contained MICA antibodies. None failed to recognize the negative serum that was included in one of the shipments. While there were important differences in the specificities of MICA antigens recognized by the different kits, the results in laboratories using the same beads were remarkably similar. Feedback was given to the participants, and especially to the producers of the antibody detection kits, after each set of four sera was sent, and results were collected. Certain beads were replaced and results improved as can be seen in the results of the third shipment of sera. It is important for laboratories to be able to accurately determine the specificity of antibodies against MICA to know whether the antibodies are donor specific. Although complete consensus was not attained, some important errors were corrected, and a better understanding of how to accurately determine MICA allele-specific antibodies was developed.
Assuntos
Antígenos HLA , Antígenos de Histocompatibilidade Classe I , Alelos , Anticorpos/sangue , Anticorpos/genética , Anticorpos/imunologia , Alemanha , Rejeição de Enxerto , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/sangue , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Transplante de RimRESUMO
Controlling the half-life of pharmaceuticals through Fc engineering is a desirable approach to achieve optimal exposure and targeting. The long serum residence time of gamma immunoglobulins is attributed to the Fc binding to the neonatal Fc receptor (FcRn). The residues in the Fc region that interact with FcRn have been mapped and individual mutations of these residues have demonstrated reduced affinity to FcRn and faster blood clearance. Here, we describe site-specific mutagenesis of Fc residues in a scFv-Fc fusion protein, as well as the mammalian production, purification, characterization, and the in vivo pharmacokinetics of these antibody fragments.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Engenharia de Proteínas/métodos , Receptores Fc/metabolismo , Soro/metabolismo , Animais , Cromatografia de Afinidade , Cromatografia em Camada Fina , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Humanos , Radioisótopos do Iodo , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores Fc/genética , Receptores Fc/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
BACKGROUND: Cancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition. METHODS: Myelomonocytic leukemic (TPH-1 and U-937) and cervical cancer (CALO and INBL) cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Student's t-test for comparison between groups. Values were considered significantly different if p < 0.05. RESULTS: THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D. CONCLUSIONS: Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.