RESUMEN
Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Ácidos Grasos Monoinsaturados/farmacología , Ácidos Grasos/farmacología , Antígenos de Histocompatibilidad Clase I , Animales , Presentación de Antígeno/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Citometría de Flujo , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Microscopía Confocal , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The functions of cholesterol and membrane microdomains in transmembrane signaling remain controversial. Edidin discusses the questions surrounding lipid rafts, membrane microdomains that have been biochemically defined but are difficult to visualize in vivo. He also discusses whether experiments showing correlation of changes in plasma membrane cholesterol with differentiation and the formation of adherens junctions in endothelial cells are consistent with a model in which lipid rafts influence the regulation of these processes.
Asunto(s)
Colesterol/fisiología , Microdominios de Membrana/fisiología , Proteínas/metabolismo , Animales , Colesterol/química , Humanos , Microdominios de Membrana/química , Fosforilación , Proteínas/químicaRESUMEN
Near-field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far-field microscopes. Near-field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near-field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.
Asunto(s)
Microscopía/métodos , Biología , Óptica y FotónicaRESUMEN
Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb(3), on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb(3) in lipid rafts with another glycosphingolipid G(M1) on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.
Asunto(s)
Transporte Biológico/fisiología , Membrana Celular/metabolismo , Microdominios de Membrana/metabolismo , Toxina Shiga I/metabolismo , Trihexosilceramidas/metabolismo , Células CACO-2 , Polaridad Celular , Colesterol/metabolismo , Transferencia de Energía , Colorantes Fluorescentes/metabolismo , Gangliósido G(M1)/metabolismo , Humanos , Microdominios de Membrana/química , Microscopía Fluorescente/métodos , Agregación de Receptores/fisiología , Receptores de Superficie Celular/metabolismoRESUMEN
Many types of experiment show that the plasma membranes of cells are patchy and locally differentiated into domains. Some of these domains seem to arise through the confinement of diffusible membrane proteins. Others might arise through lipid-lipid interactions. Both types of domain are transient on a biological timescale but both could create local conditions that enhance molecular interactions, such as those that occur in receptor-mediated signaling.
Asunto(s)
Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Difusión , HumanosRESUMEN
There is accumulating evidence that CD4(+) T cell responses are important in antitumor immunity. Accordingly, we generated CD4(+) T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4(+) hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4(+) response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4(+) epitope.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Epítopos Inmunodominantes/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Antígenos de Neoplasias/química , Linfocitos T CD4-Positivos/inmunología , Calreticulina , Retículo Endoplásmico/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Productos del Gen env/metabolismo , Calor , Hibridomas/metabolismo , Virus de la Leucemia Murina/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Ratones Transgénicos , Unión Proteica/inmunología , Conformación Proteica , Desnaturalización Proteica , Células Tumorales CultivadasRESUMEN
Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Microdominios de Membrana , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Clatrina/metabolismo , Simulación por Computador , Vesículas Citoplasmáticas/efectos de los fármacos , Dinaminas , Endocitosis/efectos de los fármacos , Fibroblastos , Fluorescencia , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Antígenos HLA-A/metabolismo , Células HeLa , Humanos , Soluciones Hipertónicas/farmacología , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Microesferas , Modelos Biológicos , Mutación , Temperatura , Factores de TiempoRESUMEN
Activation of phosphatidylinositol 3'-kinase (PI 3'-K) after ligation of CD3 protects Th2 cells from CD95-mediated apoptosis. Here we show that protection is achieved by inhibition of the formation of CD95 aggregates and consequent activation of caspase-8. Inhibition of aggregate formation is mediated by changes in the actin cytoskeleton, which in turn inhibit lateral diffusion of CD95, reducing its diffusion coefficient, D, 10-fold. After cytochalasin D treatment of stimulated cells, the lateral diffusion of CD95 increases to the value measured on unstimulated cells, and CD95 molecules aggregate to process caspase-8 and mediate apoptosis. Regulation of functional receptor formation by modulating lateral diffusion is a novel mechanism for controlling receptor activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/inmunología , Inhibidores de Caspasas , Caspasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal/inmunología , Receptor fas/metabolismo , Actinas/fisiología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Caspasa 8 , Caspasa 9 , Membrana Celular/enzimología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Clonales , Citoesqueleto/enzimología , Citoesqueleto/inmunología , Citoesqueleto/fisiología , Difusión , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Hidrólisis , Agregación de Receptores/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor fas/inmunologíaRESUMEN
Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.
Asunto(s)
Acetilcisteína/análogos & derivados , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Antígeno HLA-A2/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Acetilcisteína/farmacología , Secuencia de Aminoácidos , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Línea Celular Transformada , Inhibidores de Cisteína Proteinasa/farmacología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Transferencia de Energía/efectos de los fármacos , Transferencia de Energía/genética , Transferencia de Energía/inmunología , Productos del Gen tax/inmunología , Productos del Gen tax/metabolismo , Proteínas Fluorescentes Verdes , Antígeno HLA-A2/genética , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/inmunología , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de FluorescenciaRESUMEN
While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.
Asunto(s)
Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Colesterol/metabolismo , Reactivos de Enlaces Cruzados , Dimerización , Antígenos H-2/química , Antígenos H-2/metabolismo , Técnicas In Vitro , Cinética , Lípidos de la Membrana/metabolismo , Ratones , Modelos Moleculares , Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Receptores de Antígenos de Linfocitos T/química , Linfocitos T/metabolismoRESUMEN
"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.
Asunto(s)
Membrana Celular/química , Toxina del Cólera/análisis , Glicosilfosfatidilinositoles/análisis , Proteínas de la Membrana/análisis , Microscopía/métodos , Animales , Transferencia de Energía , Gangliósido G(M1)/análisis , Glicoesfingolípidos/análisis , Humanos , RatasRESUMEN
It has been assumed that upon dissociation from TAP, MHC class I molecules exit the ER by nonselective bulk flow. We now show that exit must occur by association with cargo receptors. Inconsistent with exit by bulk flow, loading of MHC class I molecules with high-affinity peptides triggers dissociation from TAP but has no effect on rates of ER-to-Golgi transport. Moreover, peptide-loaded MHC class I molecules accumulate at ER exit sites from which TAP molecules are excluded. Consistent with receptor-mediated exit, ER-to-Golgi transport of MHC class I molecules is independent of their cytoplasmic tails, which themselves lack ER export motifs. In addition, we show that MHC class I molecules associate with the putative cargo receptor BAP31.
Asunto(s)
Presentación de Antígeno/inmunología , Retículo Endoplásmico/inmunología , Antígenos H-2/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas de la Membrana , Animales , Transporte Biológico , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Células L , Ratones , Ratones Endogámicos C57BL , Proteínas/inmunología , Proteínas/metabolismoRESUMEN
Patches (lateral heterogeneities) of cell surface membrane proteins and lipids have been imaged by a number of different microscopy techniques. This patchiness has been taken as evidence for the organization of membranes into domains whose composition differs from the average for the entire membrane. However, the mechanism and specificity of patch formation are not understood. Here we show how vesicle traffic to and from a cell surface membrane can create patches of molecules of the size observed experimentally. Our computer model takes into account lateral diffusion, barriers to lateral diffusion, and vesicle traffic to and from the plasma membrane. Neither barriers nor vesicle traffic alone create and maintain patches. Only the combination of these produces a dynamic but persistent patchiness of membrane proteins and lipids.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Modelos Biológicos , Algoritmos , Fenómenos Biofísicos , Biofisica , Simulación por Computador , Difusión , Fluidez de la Membrana , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismoRESUMEN
Cells adjust their membrane lipid composition when they adapt to grow at different temperatures. The consequences of this adjustment for membrane properties and functions are not well understood. Our report shows that the temperature dependence of the diffusion of a probe molecule in multilayers formed from total lipid extracts of E. coli has an anomalous maximum at a temperature corresponding to the growth temperature of each bacterial preparation (25, 29, and 32 degrees C). This increase in the lateral diffusion coefficient, D, is characteristic of membrane lipids in a critical state, for which large fluctuations of molecular area in the plane of the bilayer are expected. Therefore, changes in lipid composition may be due to a requirement that cells maintain their membranes in a state where molecular interactions and reaction rates are readily modulated by small, local perturbations of membrane organization.
Asunto(s)
Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , División Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/fisiología , Difusión , Colorantes Fluorescentes/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/fisiología , Fosfatidiletanolaminas/metabolismo , Fotoquímica , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Retículo Endoplásmico/metabolismo , Antígenos H-2/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/química , Animales , Difusión , Proteínas Fluorescentes Verdes , Antígenos H-2/química , Antígeno de Histocompatibilidad H-2D , Proteínas Luminiscentes/química , Ratones , Ratones NoqueadosRESUMEN
Membrane microdomains ("lipid rafts") enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 A). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5' nucleotidase (5' NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5' NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5' NT is in clusters, the data imply that most 5' NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.
Asunto(s)
5'-Nucleotidasa/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Espectrometría de Fluorescencia , Animales , Anticuerpos/metabolismo , Carbocianinas , Línea Celular , Reactivos de Enlaces Cruzados , Detergentes , Perros , Transferencia de Energía , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Octoxinol , Ratas , Espectrometría de Fluorescencia/métodosRESUMEN
Near-field scanning optical microscopy (NSOM) uses the near-field interaction of light from a sharp fiber-optic probe with a sample of interest to image surfaces at a resolution beyond the diffraction limit of conventional optics. We used NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblasts, either dried or in buffer. A patchy distribution of a fluorescent lipid analog suggestive of lipid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluorescence photobleaching recovery measurements. Patches of fluorescent lipid analog were not spatially correlated with patches of transmembrane proteins, HLA class I molecules labeled with fluorescent antibody; the patchiness of the HLA class I molecules was on a smaller scale and was not localized to the same regions of membrane as the lipid analog. Sizes of HLA patches were deduced from a two-dimensional spatial autocorrelation analysis of NSOM images that resolved patches with radii of approximately 70 and approximately 600 nm on fixed, dried cells labeled with IgG and 300-600 nm on cells labeled with Fab and imaged in buffer. The large-size patches were also resolved by far-field microscopy. Both the spatial autocorrelation analysis and estimates from fluorescence intensity indicate that the small patches seen on fixed, dried cells contain approximately 25-125 HLA-I molecules each.
Asunto(s)
Membrana Celular/ultraestructura , Línea Celular , Fibroblastos/ultraestructura , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Proteínas de la Membrana/análisis , Microscopía Confocal/métodos , Modelos Estructurales , Piel/ultraestructuraRESUMEN
The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B*3501 and HLA-B*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B*3501 and -B*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex.