RESUMO
Recent advances in our understanding of biosynthesis and assembly of MHC subunits are discussed. Intracellular traffic of MHC proteins is reviewed in the context of antigen presentation. While the overall picture of antigen presentation is now clear, much of the detail of the early stages of assembly of MHC products remains to be established. The degradative pathways that result in the peptides presented by MHC molecules (in particular those serving Class I molecules) have not yet been identified with certainty.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Animais , Transporte Biológico , Endocitose , Endopeptidases/metabolismo , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade/imunologia , Humanos , Modelos Biológicos , Estrutura Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The MHC class II-associated invariant chain interacts in trimeric form with class II molecules, inhibits peptide binding, and mediates targeting of class II molecules to endosomal compartments. To dissect the different functions of the invariant (Ii) chain, a set of cDNAs, encoding truncated forms of the Ii chain, was constructed. mRNAs, transcribed from these cDNAs were translated in vitro, together with mRNAs encoding class II HLA DR1 alpha and beta subunits. An Ii chain truncation that contains the 104 NH2-terminal amino acids was able to associate with class II molecules. This construct contains the region from which class II-associated Ii chain peptides (CLIP, amino acids 81-104) are derived. The absence of a further eight residues at the COOH terminus results in a construct of 96 amino acids that is unable to associate with class II molecules. Association of the truncated Ii chains with class II molecules showed a strict correlation with inhibition of peptide binding. Removal of the NH2-terminal cytoplasmic tail and transmembrane region of Ii chain and its replacement with a cleavable signal sequence led to aberrant folding and impaired association with class II molecules. The region between amino acids 163 and 183 was found to be essential for visualization of Ii chain homotrimers by covalent cross-linking.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Coelhos , Transcrição GênicaRESUMO
The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-alpha, -beta and CD3-gamma, -delta, -epsilon, and -zeta subunits. CD3-epsilon, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-gamma or -delta, led to the expression of mainly monomeric and correctly folded epsilon subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-epsilon in a glycan-independent manner. A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon. We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.
Assuntos
Biossíntese de Proteínas/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Sequência de Aminoácidos , Complexo CD3/genética , Complexo CD3/metabolismo , Complexo CD3/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Dissulfetos/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Oxirredução , Peptídeos/genética , Peptídeos/imunologia , Polímeros/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/imunologia , Sinais Direcionadores de Proteínas/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/químicaRESUMO
Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and beta 2-microglobulin, and later, upon disintegration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2K(b) heavy chain which are obscured upon proper folding and subsequent assembly with beta 2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The K(b) heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains. At the cell surface, a pool of free K(b) heavy chains appears after 60-120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free heavy chains that arise at the cell surface are destroyed after internalization.
Assuntos
Anticorpos Monoclonais , Antígenos H-2/química , Antígenos H-2/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Linhagem Celular Transformada , Membrana Celular/imunologia , Epitopos/análise , Antígenos H-2/biossíntese , Linfoma , Substâncias Macromoleculares , Camundongos , Desnaturação Proteica , Dobramento de Proteína , Microglobulina beta-2/metabolismoRESUMO
The mechanisms that regulate developmental control of the expression of MHC class I genes during generation of extra-embryonic tissues are largely unknown. In the present study, we studied the levels of transcripts of the human HLA-B27 gene in extra-embryonic tissues of transgenic mice containing the HLA-B27 (heavy chain) gene by in situ hybridization with biotinylated single-stranded RNA probes. In contrast to extra-embryonic stromal cells and embryonic tissues which contain (varying levels of) messenger RNA coding for HLA-B27, specific transcripts were not detected in labyrintho-, or spongiotrophoblast, nor in trophoblastic giant cells. These cells are devoid of HLA A and B locus class I transcripts in man. Regulation of expression of human MHC class I genes in extra-embryonic trophoblast in transgenic animals is thus under conserved selective pressure that is retained across a species barrier. Thus, in extra-embryonic tissues, regulation of expression of MHC class I genes is distinct from the mechanisms operating in developing embryonic cells.
Assuntos
Antígenos HLA-B/genética , Complexo Principal de Histocompatibilidade , Camundongos Transgênicos/embriologia , Trofoblastos/imunologia , Animais , Regulação da Expressão Gênica , Antígeno HLA-B27 , Camundongos , Camundongos Transgênicos/genética , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Especificidade da EspécieRESUMO
Antisera have been prepared against the separated glycoprotein chains (p29 and p34) of HLA-DR antigens (p29,34) isolated from membranes of the B lymphoblastoid cell line JY (DRw4,6). These antisera (anti-p29 and anti-p34) were characterized by immunoprecipitation of in vivo labeled, detergent-solubilized extracts of JY cells grown in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. Anti-p29 and anti-p34 specifically immunoprecipitated the precursors to p29 and p34, respectively, from the cell-free translation products of a rabbit reticulocyte lysate system supplemented with polyadenylic acid-containing messenger RNA (mRNA) isolated from JY cells. These precursors, pre-p34 and two pre-p29s, appeared to be 1,500-3,000 daltons larger than their counterparts from tunicamycin-treated cells and presumably were synthesized with N-terminal extensions (signal sequences). Processing of pre-p29 and pre-p34 occurred during cell-free translation in the presence of dog pancreatic microsomes, which resulted in cleavage of the polypeptide, addition of glycan moieties, and segregation of the two chains in the cisternal portion of the microsome. the precursors of p29 and p34 were not appreciably different in isoelectric points on two-dimensional gels from p26 and p28, the light and heavy chains from antigens arise from separate mRNA, and not as a single polypeptide, which is endoproteolytically cleaved.
Assuntos
Antígenos de Histocompatibilidade Classe II/biossíntese , Biossíntese de Proteínas , Linfócitos B/metabolismo , Linhagem Celular , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Microssomos/metabolismo , Peso Molecular , RNA Mensageiro/genéticaRESUMO
Human cytomegalovirus downregulates the expression of human class I major histocompatibility complex (MHC) molecules by accelerating destruction of newly synthesized class I heavy chains. The HCMV genome contains at least two genes, US11 and US2, each of which encode a product sufficient for causing the dislocation of newly synthesized class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol. Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules. Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized. The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.
Assuntos
Alelos , Citomegalovirus/genética , Complexo Principal de Histocompatibilidade/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Glicosilação , Antígenos H-2/metabolismo , Hidrólise , Camundongos , Proteínas de Ligação a RNA/genética , Células Tumorais Cultivadas , Proteínas Virais/genéticaRESUMO
Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Catepsina D/fisiologia , Catepsinas/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/fisiologia , Sequência de Aminoácidos , Animais , Cricetinae , Inibidores de Cisteína Proteinase/farmacologia , Leupeptinas/farmacologia , Camundongos , Dados de Sequência Molecular , Polimorfismo Genético , CoelhosRESUMO
Class I major histocompatibility complex molecules require both beta 2-microglobulin (beta 2m) and peptide for efficient intracellular transport. With the exception of H-2Db and Ld, class I heavy chains have not been detectable at the surface of cells lacking beta 2m. We show that properly conformed class I heavy chains can be detected in a terminally glycosylated form indicative of cell surface expression in H-2b, H-2d, and H-2s beta 2m-/- concanavalin A (Con A)-stimulated splenocytes incubated at reduced temperature. Furthermore, we demonstrate the presence of Kb molecules at the surface of beta 2m-/- cells cultured at 37 degrees C. The mode of assembly of class I molecules encompasses two major pathways: binding of peptide to preformed "empty" heterodimers, and binding of peptide to free heavy chains, followed by recruitment of beta 2m. In support of the existence of the latter pathway, we provide evidence for a role of peptide in intracellular transport of free class I heavy chains, through analysis of Con A-stimulated splenocytes from transporter associated with antigen processing 1 (TAP1)-/-, beta 2m-/-, and double-mutant TAP1/beta 2m-/- mice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Dobramento de Proteína , Microglobulina beta-2/fisiologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Transporte Biológico , Citometria de Fluxo , Antígenos H-2/genética , Antígenos de Histocompatibilidade Classe I/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Temperatura , Microglobulina beta-2/análiseRESUMO
US11 and US2 encode gene products expressed early in the replicative cycle of human cytomegalovirus (HCMV), which cause dislocation of human and murine major histocompatibility complex (MHC) class I molecules from the lumen of the endoplasmic reticulum to the cytosol, where the class I heavy chains are rapidly degraded. Human histocompatibility leukocyte antigens (HLA)-C and HLA-G are uniquely resistant to the effects of both US11 and US2 in a human trophoblast cell line as well as in porcine endothelial cells stably transfected with human class I genes. Dislocation and degradation of MHC class I heavy chains do not appear to involve cell type-specific factors, as US11 and US2 are fully active in this xenogeneic model. Importantly, trophoblasts HLA-G and HLA-C possess unique characteristics that allow their escape from HCMV-associated MHC class I degradation. Trophoblast class I molecules could serve not only to block recognition by natural killer cells, but also to guide virus-specific HLA-C- and possibly HLA-G-restricted cytotoxic T-lymphocytes to their targets.
Assuntos
Citomegalovirus/imunologia , Antígenos HLA/biossíntese , Antígenos HLA-C/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Trofoblastos/metabolismo , Animais , Linhagem Celular Transformada , Citomegalovirus/genética , Endotélio Vascular , Antígenos H-2/imunologia , Antígenos HLA/genética , Antígenos HLA-C/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Suínos , Trofoblastos/imunologia , Células Tumorais Cultivadas , Proteínas Virais/genética , Proteínas Virais/imunologia , Microglobulina beta-2/imunologiaRESUMO
The set of peptides that is bound by a given major histocompatibility complex class I product can be described by one or two properly spaced anchor residues, and two properly spaced peptide termini, approximately 8-10 residues apart. Using radiolabeled peptide libraries, we examined whether mutations in those "pockets" in class I Kb molecules that do not seem critically involved in the interaction with the peptide anchor residues, do exert an effect on the set of preferred peptides. We find that mutations in all the pockets found in the structure of Kb have a significant effect on the peptide preference of the molecule, and their recognition by cytotoxic T cells. Alterations in substrate specificity are also observed for mutations involving residues that interact with main chain atoms in both peptide termini. These findings challenge a static view of the interaction of peptide termini with their respective pockets in the class I molecule, and imply a role for the minor pockets in peptide selectivity.
Assuntos
Antígenos H-2/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Antígenos H-2/química , Antígenos H-2/genética , Camundongos , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Polimorfismo Genético , Relação Estrutura-AtividadeRESUMO
The B6 anti-bm6 allospecific CTL response is strictly dependent on CD4+ cells when using LPS blasts as stimulator cells. Altering the N-linked carbohydrates on stimulator cells by use of the N-linked trimming glycosidase inhibitors 1-deoxymannojirimycin and swainsonine, or by treatment with bacterial neuraminidase, results in a restoration of the B6 anti-bm6 response in the absence of CD4+ cells. The extent of restoration is inversely correlated with the number of sialic acids present on N-linked glycans of stimulator cells.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Polissacarídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Sequência de Carboidratos , Eletroforese em Gel de Poliacrilamida , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
The human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain beta 2-microglobulin complexes is observed for both HLA-A2 and HLA-B5. Culture at reduced temperatures (26 or 20 degrees C) does not increase the amount of class I molecules transported, unlike what has been reported for the class I assembly-deficient mouse mutant cell line RMA-S. The HLA-B5 and the HLA-A2 complexes formed by T2 are thermolabile in cell lysates, albeit to different degrees. The thermolability of HLA-B5 can be overcome by addition of HLA-B5-presentable peptides, obtained by trifluoroacetic acid extraction from an HLA-B5-positive cell line, underlining the necessity of peptide for class I stability and indicating that T2-derived class I complexes are devoid of peptide. Cytoplast fusion of T2 cells with RMA-S cells shows the defect in class I assembly of RMA-S to be similar to that of T2. Localization of class I molecules observed by immuno-electron microscopy reveals the accumulation in the T2 cell line of both HLA-B5 and HLA-A2 in the endoplasmic reticulum (ER). Class I molecules are present in all the cisternae of the Golgi complex of T2, but the ratio of HLA-A and -B locus products in the Golgi area differs significantly from that at the cell surface. We conclude that the requirement for peptide in transport of class I molecules manifests itself at a stage beyond the ER, most likely the Golgi area.
Assuntos
Antígenos de Histocompatibilidade Classe I/análise , Linhagem Celular , Antígeno HLA-A2/análise , Antígenos HLA-B/análise , Humanos , Microscopia Imunoeletrônica , Temperatura , Microglobulina beta-2/análiseRESUMO
In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II- and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Antígenos/metabolismo , Linfócitos B/imunologia , Endossomos/imunologia , Antígenos HLA-D/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/ultraestrutura , Antígenos/química , Linfócitos B/ultraestrutura , Transporte Biológico , Compartimento Celular , Linhagem Celular , Retículo Endoplasmático/imunologia , Complexo de Golgi/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Microscopia Eletrônica , Peptídeos/imunologia , Peptídeos/metabolismoRESUMO
The c-myc oncogene downregulates class I HLA expression in human melanoma. The major class I HLA antigens are encoded by three loci, A, B, and C, and we investigated whether these loci are suppressed equally by c-myc. In three melanoma cell lines with high c-myc expression, we analyzed mRNA, protein, and cell surface expression of the class I HLA antigens. Whereas the HLA-B locus expression was found to be strongly reduced, the HLA-A locus was expressed normally. Analysis of c-myc-transfected clones of two melanoma cell lines confirmed that c-myc preferentially suppresses the class I HLA-B locus. Immunohistochemical analysis of fresh melanoma lesions also showed that in the tumors the HLA-A loci are expressed normally, while on the majority of tumor cells no HLA-B antigen expression was found. This downregulation may have consequences for the recognition of malignant cells by tumor-infiltrating lymphocytes. Our results predict that HLA-B-restricted cytotoxic T cells will be unable to kill high c-myc-expressing melanoma cells.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Melanoma/genética , Proto-Oncogenes , Alelos , Animais , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Melanoma/imunologia , Camundongos , RNA Mensageiro/análise , Linfócitos T Citotóxicos/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2-35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas Imediatamente Precoces/química , Simplexvirus/patogenicidade , Proteínas Virais , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Humanos , Substâncias Macromoleculares , Complexo Principal de Histocompatibilidade , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The transporter associated with antigen processing (TAP) delivers peptides to the lumen of the endoplasmic reticulum in an adenosine triphosphate (ATP) dependent fashion for presentation by major histocompatibility complex class I molecules. We show that the mouse TAP translocator (H-2b haplotype) selects peptides based on a minimal size of nine residues, and on the presence of a hydrophobic COOH-terminal amino acid. The preponderance of COOH-terminal hydrophobic amino acids in peptides capable of binding to mouse class I molecules thus fits remarkably well with the specificity of the TAP translocator. In addition to transport in the lumenal direction, efflux of peptide in the cytosolic direction is observed in an ATP- and temperature-dependent manner. By maintaining a low peptide concentration at the site of class I assembly, this efflux mechanism may ensure that class I molecules are loaded preferentially with high affinity peptides.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Transporte Biológico , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , TemperaturaRESUMO
Malignant tumor cells can escape CD8+ cytotoxic T cell killing by downregulating class I major histocompatibility complex (MHC) expression. Stable class I MHC surface expression requires loading of the heavy chain/light chain dimer with antigenic peptide, which is delivered to class I MHC molecules in the endoplasmic reticulum by the presumed peptide transporter, encoded by the transporter associated with antigen presentation (TAP) 1 and 2 genes. We have investigated whether loss of class I MHC expression frequently observed in different cancers could result from interference with TAP function. A polyclonal antiserum, raised against a bacterial glutathione S-transferase/human TAP-1 fusion protein, was used for the immunohistochemical analysis of TAP-1 expression in 76 cervical carcinomas. Results showed loss of TAP-1 expression in neoplastic cells in 37 out of 76 carcinomas. Immunohistochemical double staining procedures in combination with HLA-specific antibodies revealed congruent loss at the single cell level of TAP-1 and HLA-A/B expression in 28 out of 37 carcinomas. The remaining samples expressed HLA(-A) in the absence of TAP-1 (n = 6) or showed loss of HLA(-A/B) while TAP-1 was expressed (n = 3). These data strongly indicate that inhibition of peptide transport by downregulation of TAP-1 is a potential strategy of malignant cells to evade immune surveillance.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/metabolismo , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe II/metabolismo , Neoplasias do Colo do Útero/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Especificidade de Anticorpos , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Soros ImunesRESUMO
The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Catepsinas/imunologia , Endopeptidases , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos Alveolares/imunologia , Animais , Catepsina F , Catepsina L , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Cricetinae , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Expressão Gênica , Humanos , Imunoglobulina E/imunologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/imunologia , Sulfonas/farmacologiaRESUMO
Mice harboring a deletion of the gene encoding the transporter associated with antigen presentation-1 (TAP1) are impaired in providing major histocompatibility complex (MHC) class I molecules with peptides of cytosolic origin and lack stable MHC class I cell surface expression. They consequently have a strongly reduced number of CD8+ T cells. To examine whether selection of CD8+ T cells is dependent on TAP-dependent peptides, we partially restored MHC class I cell surface expression in TAP1-deficient mice by introduction of human beta 2-microglobulin. We show that selection of functional CD8+ T cells can be augmented in vivo in the absence of TAP1-dependent peptides.