RESUMEN
We have cloned and sequenced the predominant germline V kappa gene segment expressed by B cells of strain A origin that synthesize antibodies with specificity for Ars. In hybridomas synthesizing anti-Ars antibodies, this V kappa gene segment (V kappa IdCR) has been found exclusively associated with the J kappa 1 gene segment without exhibiting junctional sequence variation. Sequence comparisons of the germline V kappa IdCR gene with expressed derivatives reveals that the latter frequently contain somatically introduced amino acid replacements. Taken together with results of previous structural analyses, these results show that the predominant population of IdCR+ V regions elicited in the secondary immune response is encoded by one or two combinations of V gene segments, has little junctional diversity, and is extensively diversified by somatic mutation in both heavy and light chains.
Asunto(s)
Anticuerpos Monoclonales/genética , Compuestos Azo/inmunología , Inmunización , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , p-Azobencenoarsonato/inmunología , Animales , Clonación Molecular , Enzimas de Restricción del ADN , ADN Recombinante , Desoxirribonucleasa BamHI , Hibridomas/inmunología , Ratones , Ratones Endogámicos A , Hibridación de Ácido NucleicoRESUMEN
Major histocompatibility complex (MHC) class I molecules bind peptides that are delivered from the cytosol into the endoplasmic reticulum by the MHC-encoded transporter associated with antigen processing (TAP). Peptide capture by immature heterodimers of class I heavy chains and beta 2-microglobulin may be facilitated by their physical association with TAP. A genetic defect in a human mutant cell line causes the complete failure of diverse class I heterodimers to associate with TAP. This deficiency impairs the ability of the class I heterodimers to efficiently capture peptides and results from loss of function of an unidentified gene or genes linked to the MHC.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Complejo Mayor de Histocompatibilidad , Péptidos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/metabolismo , Calnexina , Línea Celular , Retículo Endoplásmico/metabolismo , Antígenos HLA/metabolismo , Antígeno HLA-A1/metabolismo , Antígenos HLA-B/metabolismo , Antígeno HLA-B8/metabolismo , Antígenos HLA-G , Humanos , Ligandos , Complejo Mayor de Histocompatibilidad/genética , Datos de Secuencia Molecular , Mutación , Transfección , Microglobulina beta-2/metabolismoRESUMEN
Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antiportadores/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Secuencia de Aminoácidos , Presentación de Antígeno , Antiportadores/química , Antiportadores/genética , Proteínas de Unión al Calcio/metabolismo , Calreticulina , Línea Celular , Línea Celular Transformada , Mapeo Cromosómico , Cromosomas Humanos Par 6 , Clonación Molecular , Dimerización , Retículo Endoplásmico/metabolismo , Ligamiento Genético , Humanos , Inmunoglobulina G/química , Inmunoglobulinas/química , Inmunoglobulinas/genética , Complejo Mayor de Histocompatibilidad/genética , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Ribonucleoproteínas/metabolismo , Homología de Secuencia de Aminoácido , Linfocitos T Citotóxicos , Células Tumorales CultivadasRESUMEN
A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Proteínas de Transporte de Monosacáridos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética , Cromatografía de Afinidad , Proteínas de Unión a Maltosa , Plásmidos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
The cDNA synthesized from mRNA of Dirofilaria immitis female adult worms was cloned into the expression vector lambda gt11. Screening the library with a hyperimmune rabbit antiserum raised against adult worm homogenates yielded several antigen positive clones. One of these clones, lambda cDi2, was recognized by rabbit antisera raised against either D. immitis L-3, adult, Brugia malayi L-3 or Onchocerca volvulus adult worm antigen, as well as by antisera from humans naturally infected with O. volvulus or Wuchereria bancrofti. Affinity-purified anti-lambda cDi2 antibodies reacted with a 97-kDa protein on Western transfers of adult D. immitis antigen extracts that were reduced with beta-mercaptoethanol. The whole rabbit anti-D. immitis adult antiserum depleted of anti-lambda cDi2 antibodies exhibited decreased reactivity to this 97-kDa band. A monoclonal antibody (IA6) that specifically binds Schistosoma mansoni paramyosin also recognised a 97-kDa protein in D. immitis extracts upon Western transfer. The deduced amino acid sequence of partial DNA sequence from lambda cDi2 showed some similarity to nematode myosin, and gave a stretch of 82 amino acids that is 91.5% identical to Caenorhabditis elegans paramyosin: thus, lambda cDi2 encodes D. immitis paramyosin.
Asunto(s)
Antígenos Helmínticos/genética , Dirofilaria/genética , Filarioidea/genética , Proteínas Recombinantes/genética , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Helmínticos/inmunología , Bacteriófago lambda/genética , Secuencia de Bases , Clonación Molecular/métodos , Reacciones Cruzadas , ADN Recombinante , Dirofilaria/crecimiento & desarrollo , Femenino , Immunoblotting , Lisogenia , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Especificidad de la Especie , Tropomiosina/biosíntesis , Tropomiosina/inmunologíaRESUMEN
Single-residue changes were introduced into the murine major histocompatibility complex class I molecule H-2Kb at positions 65 and 69, which are predicted to point up from the alpha-helix of the alpha 1 domain and not into the peptide binding groove. Mutated and wild-type genes were transfected into the murine cell line P815 (H-2d). We present evidence that the changes did not affect the binding of three foreign peptides that are recognized by cytotoxic T lymphocytes (CTL) in association with H-2Kb. Additionally, the mutants provoked strong alloreactive responses in T cells from mice expressing unmutated H-2Kb. The alloreactive CTL were specific for self peptides, which could be extracted from wild-type H-2Kb molecules, recognized in the context of the mutant class I.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos H-2/fisiología , Isoantígenos/inmunología , Complejo Mayor de Histocompatibilidad , Animales , Antígenos Virales/inmunología , Antígenos H-2/química , Ratones , Ratones Endogámicos , Mutagénesis Sitio-Dirigida , Ovalbúmina/inmunología , Relación Estructura-Actividad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The stable assembly of MHC class I molecules with peptides in the endoplasmic reticulum (ER) involves several accessory molecules. One of these accessory molecules is tapasin, a transmembrane protein that tethers empty class I molecules to the peptide transporter associated with antigen processing (TAP). Here, evidence is presented that tapasin retains class I molecules in the ER until they acquire high-affinity peptides.
Asunto(s)
Presentación de Antígeno/inmunología , Antiportadores/inmunología , Retículo Endoplásmico/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Chaperonas Moleculares/inmunología , Péptidos/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/inmunología , Animales , Humanos , Proteínas de Transporte de MembranaRESUMEN
A transgenic mouse has been made that expresses a mutant MHC class I H-2Kb molecule with glutamic acid at position 65 (E65) in place of glutamine. The side chain at position 65, on the outward face of the alpha-helix of the alpha 1 domain of the class I molecule, interacts with the TCR, and not with the peptide binding groove. The transgenic mouse, on a DBA/2 background, mounts Kb,E65-restricted Ag-specific responses to conventional Kb-restricted Ag such as OVA and vesicular stomatitis virus, and shows strong alloreactivity to wild-type Kb. The transgenic mouse also mounts a primary in vitro alloreactive response directed to a mutant molecule with aspartic acid at position 65 (D65). This response is relatively weak, probably because of the structural similarities between aspartic and glutamic acid side chains; both have carboxylic termini, and the aspartic acid side chain is shorter by a single secondary carbon. The alloreactive CTL lines elicited by this conservative change are cross-reactive among several position-65 variants of H-2Kb. Individual CTL clones are specific for self peptides that can be extracted from cells expressing Kb,E65, and from purified wild-type Kb molecules, and that are recognized in the context of the D65 residue. Thus, the smallest variance from self in a class I molecule, even outside the peptide binding groove, can be antigenic.
Asunto(s)
Antígenos H-2/inmunología , Fragmentos de Péptidos/inmunología , Animales , Línea Celular , Reacciones Cruzadas , Antígenos H-2/química , Antígenos H-2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Mutación , Relación Estructura-Actividad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The T cell receptor (TcR) on CD8+ T lymphocytes recognizes a complex which consists of a major histocompatibility complex (MHC) heavy chain, beta 2-microglobulin (beta 2M), and peptide on the surface of antigen-presenting cells. Mutational analyses have suggested that the TcR recognizes both the alpha 1 and alpha 2 domains of the heavy chain as well as the peptide. In light of this, it is of interest to know to what extent the heavy chain domains take on distinct conformations when bound to individual peptides. It has recently been shown that antibodies which recognize the Kb MHC complex are sensitive to which peptides are bound in the groove. We have extended this analysis to include eight Kb-specific antibodies, seven of which are peptide sensitive. These antibodies, all of which are allo-antibodies, recognize Kb-bearing cells which, it is now appreciated, have a highly heterogeneous mix of self peptides presented in their grooves. We show that these self peptides also can affect antibody binding. It has been suggested that peptides alter the conformation of the alpha 1 and alpha 2 domains of the heavy chain and that this in turn affects the recognition of Kb by antibody. An alternative hypothesis is that solvent-exposed peptide side chains may prevent the antibody from binding the complex. Using a panel of 128 single-amino acid variants of a Kb-binding antigenic peptide from ovalbumin we show that for most Kb-specific antibodies, the second idea is more likely. Those variants which prevent antibody binding are at solvent exposed positions, and in general, the bulkier the side chain, the greater the inhibition of antibody binding. However, in the case of two antibodies, 100.30 and 34.4.20, the peptide residues which affect antibody recognition are buried, suggesting that these antibodies see an alternate conformation of the peptide/MHC complex.
Asunto(s)
Anticuerpos/metabolismo , Variación Genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Oligopéptidos/genética , Oligopéptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Línea Celular , Antígenos H-2/química , Antígenos H-2/metabolismo , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Oligopéptidos/metabolismoRESUMEN
T lymphocytes recognize antigens as peptide fragments associated with molecules encoded by the major histocompatibility complex (MHC) and expressed on the surface of antigen-presenting cells. In the thymus, T cells bearing alpha beta receptors that react with the MHC molecules expressed by radioresistant stromal elements are positively selected for maturation. In (A x B-->A) bone marrow chimaeras, T cells restricted to the MHC-A haplotype are positively selected, whereas MHC-B-reactive thymocytes are not. We investigated whether the introduction of particular thymic stromal elements bearing MHC-B molecules could alter the fate of B-reactive T cells in these (A x B-->A) chimaeras. Thymic epithelial cell (TEC) lines expressing H-2b were introduced by intrathymic injection into (H-2b/s-->H2s) bone marrow chimaeras and we measured their ability to generate H-2b-restricted cytotoxic T-lymphocytes (CTLs). We report here that one TEC line, 427.1, was able positively to select CTLs specific for influenza and vesicular stomatitis virus antigens in association with class I H-2b molecules. In addition, line 427.1 can process cytoplasmic proteins for presentation to H-2Kb- and H-2Db-restricted CTLs. Thus, a TEC line capable of normal class I MHC antigen processing and presentation in vitro can induce positive selection after intrathymic injection.
Asunto(s)
Antígenos H-2/inmunología , Linfocitos T Citotóxicos/inmunología , Timo/citología , Animales , Antígenos Virales/inmunología , Secuencia de Bases , Citotoxicidad Inmunológica , Inmunidad Celular , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Orthomyxoviridae/inmunología , Quimera por Radiación , Células Tumorales Cultivadas , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.
Asunto(s)
Epítopos/inmunología , Antígeno H-Y/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Biblioteca Genómica , Antígenos de Histocompatibilidad Clase I/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and beta 2-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP.
Asunto(s)
Antiportadores/metabolismo , Proteínas Portadoras/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Inmunoglobulinas/metabolismo , Línea Celular , Cromosomas Humanos Par 6 , Dimerización , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Proteínas de Transporte de MembranaRESUMEN
Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products.
Asunto(s)
Antiportadores/genética , Ligamiento Genético , Antígenos H-2/genética , Antígenos de Histocompatibilidad Clase I/biosíntesis , Inmunoglobulinas/genética , Secuencia de Aminoácidos , Animales , Antiportadores/inmunología , Antiportadores/fisiología , Línea Celular , Mapeo Cromosómico , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/fisiología , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.
Asunto(s)
Antígenos Helmínticos/genética , Dirofilaria immitis/inmunología , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/metabolismo , Secuencia de Bases , Clonación Molecular , Dirofilaria immitis/genética , Dirofilaria immitis/ultraestructura , Genes , Proteínas del Helminto/inmunología , Inmunohistoquímica , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAP(o)) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAP(o) mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAP(o) cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Antiportadores/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoglobulinas/fisiología , N-Formilmetionina/metabolismo , Péptidos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Presentación de Antígeno , Antiportadores/genética , Unión Competitiva/inmunología , Línea Celular Transformada , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Inmunoglobulinas/deficiencia , Inmunoglobulinas/genética , Sustancias Macromoleculares , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Células Tumorales Cultivadas , Microglobulina beta-2/metabolismoRESUMEN
Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.
Asunto(s)
Presentación de Antígeno , Antiportadores/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/genética , Antígenos/metabolismo , Línea Celular , Dimerización , Drosophila melanogaster , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Genes MHC Clase I , Proteínas de Transporte de Membrana , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Ovalbúmina/genética , Ovalbúmina/inmunología , Conformación Proteica , TransfecciónRESUMEN
H2-M3 is a class Ib MHC molecule that binds a highly restricted pool of peptides, resulting in its intracellular retention under normal conditions. However, addition of exogenous M3 ligands induces its escape from the endoplasmic reticulum (ER) and, ultimately, its expression at the cell surface. These features of M3 make it a powerful and novel model system to study the potentially interrelated functions of the ER-resident class I chaperone tapasin. The functions ascribed to tapasin include: 1) ER retention of peptide-empty class I molecules, 2) TAP stabilization resulting in increased peptide transport, 3) direct facilitation of peptide binding by class I, and 4) peptide editing. We report in this study that M3 is associated with the peptide-loading complex and that incubation of live cells with M3 ligands dramatically decreased this association. Furthermore, high levels of open conformers of M3 were efficiently retained intracellularly in tapasin-deficient cells, and addition of exogenous M3 ligands resulted in substantial surface induction that was enhanced by coexpression of either membrane-bound or soluble tapasin. Thus, in the case of M3, tapasin directly facilitates intracellular peptide binding, but is not required for intracellular retention of open conformers. As an alternative approach to define unique aspects of M3 biosynthesis, M3 was expressed in human cell lines that lack an M3 ortholog, but support expression of murine class Ia molecules. Unexpectedly, peptide-induced surface expression of M3 was observed in only one of two cell lines. These results demonstrate that M3 expression is dependent on a unique factor compared with class Ia molecules.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antiportadores/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/química , Inmunoglobulinas/fisiología , Péptidos/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adyuvantes Inmunológicos/deficiencia , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Animales , Antiportadores/deficiencia , Antiportadores/genética , Antiportadores/metabolismo , Línea Celular Transformada , Epítopos/química , Epítopos/genética , Epítopos/metabolismo , Antígenos H-2/metabolismo , Células HeLa , Antígeno de Histocompatibilidad H-2D , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas/deficiencia , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Células L , Proteínas de Transporte de Membrana , Ratones , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Conformación Proteica , TransfecciónRESUMEN
Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.