Major Histocompatibility Complex Class I Proteins Research Articles

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide-empty intermediate.

The cell biology and biochemistry of peptide exchange on major histocompatibility complex class I (MHC-I) proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross-presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC-I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction. We find that low pH stabilizes the peptide-empty forms of MHC-I that occur as intermediates of the exchange reaction, and that is synergizes with dipeptides and with disulfide-mediated stabilization of MHC-I.

How ERAP1 and ERAP2 Shape the Peptidomes of Disease-Associated MHC-I Proteins.

Four inflammatory diseases are strongly associated with Major Histocompatibility Complex class I (MHC-I) molecules: birdshot chorioretinopathy (HLA-A*29:02), ankylosing spondylitis (HLA-B*27), Behçet's disease (HLA-B*51), and psoriasis (HLA-C*06:02). The endoplasmic reticulum aminopeptidases (ERAP) 1 and 2 are also risk factors for these diseases. Since both enzymes are involved in the final processing steps of MHC-I ligands it is reasonable to assume that MHC-I-bound peptides play a significant pathogenetic role. This review will mainly focus on recent studies concerning the effects of ERAP1 and ERAP2 polymorphism and expression on shaping the peptidome of disease-associated MHC-I molecules in live cells. These studies will be discussed in the context of the distinct mechanisms and substrate preferences of both enzymes, their different patterns of genetic association with various diseases, the role of polymorphisms determining changes in enzymatic activity or expression levels, and the distinct peptidomes of disease-associated MHC-I allotypes. ERAP1 and ERAP2 polymorphism and expression induce significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and, without excluding a degree of functional inter-dependence between both enzymes, reflect largely separate roles in their processing of MHC-I ligands. The studies reviewed here provide a molecular basis for the distinct patterns of genetic association of ERAP1 and ERAP2 with disease and for the pathogenetic role of peptides. The allotype-dependent alterations induced on distinct peptidomes may explain that the joint association of both enzymes and unrelated MHC-I alleles influence different pathological outcomes.

Changes in Expressions of Major Histocompatibility Complex Class I, Paired-Immunoglobulin-Like Receptor B, and Cluster of Differentiation 3ζ in Motor Cortical Representations of the Brachial Plexus After Avulsion in Rats

Major histocompatibility complex class I (MHCI), paired-immunoglobulin-like receptor B (PirB), and cluster of differentiation 3ζ (CD3ζ) negatively regulate neuronal plasticity in developing and adult brains. The aim of this study was to evaluate expressive changes of these factors in motor cortical representations of the brachial plexus (MCRBP) after total brachial plexus root avulsion (tBPRA). A total of 45 rats were randomly and equally divided into 3 groups for evaluating mRNA and protein expression levels of MHCI, PirB, and CD3ζ: 7 days, 3 months, and control. In the 7-day and 3-month groups, expressions were examined at 7 days and 3 months, respectively, after left tBPRA. In the control group, the brachial plexus was uninjured. Three rats from each group were used for examining expressions of MHCI, PirB, and CD3ζ proteins by immunofluorescence labeling, 6 rats for quantification of MHCI, PirB, and CD3ζ mRNAs by real-time quantitative polymerase chain reaction, and the remaining 6 animals for quantification of MHCI, PirB, and CD3ζ proteins by Western blotting. In the original MCRBP, mRNA and protein expression levels of MHCI, PirB, and CD3ζ were down-regulated 7 days postinjury compared with control (P<0.01). Interestingly, mRNA and protein expression levels of these factors were up-regulated at 3 months compared with 7 days (P < 0.01), excepting PirB protein, whose expression was not increasing (P > 0.05). Recovery of protein expressions were initiated from near the border region of the original MCRBP. MHCI, PirB, and CD3ζ may participate in motor cortical reorganization after tBPRA.

Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

BackgroundMajor histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown.ResultsHere we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI’s cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction.ConclusionsThese results identify cryptic protein interaction motifs in the cytoplasmic domain of MHCI. In so doing, they suggest that the cytoplasmic domain of MHCI could participate in previously unsuspected PDZ mediated protein-protein interactions at neuronal as well as immunological synapses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0154-z) contains supplementary material, which is available to authorized users.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane.

Expression and alternative splicing of classical and nonclassical MHCI genes in the hippocampus and neuromuscular junction.

The major histocompatibility complex class I (MHCI) is a large gene family, with over 20 members in mouse. Some MHCIs are well-known for their critical roles in the immune response. Studies in mice which lack stable cell-surface expression of many MHCI proteins suggest that one or more MHCIs also play unexpected, essential roles in the establishment, function, and modification of neuronal synapses. However, there is little information about which genes mediate MHCI's effects in neurons. In this study, RT-PCR was used to simultaneously assess transcription of many MHCI genes in regions of the central and peripheral nervous system where MHCI has a known or suspected role. In the hippocampus, a part of the CNS where MHCI regulates synapse density, synaptic transmission, and plasticity, we found that more than a dozen MHCI genes are transcribed. Single-cell RT-PCR revealed that individual hippocampal neurons can express more than one MHCI gene, and that the MHCI gene expression profile of CA1 pyramidal neurons differs significantly from that of CA3 pyramidal neurons or granule cells of the dentate gyrus. MHCI gene expression was also assessed at the neuromuscular junction (NMJ), a part of the peripheral nervous system (PNS) where MHCI plays a role in developmental synapse elimination, aging-related synapse loss, and neuronal regeneration. Four MHCI genes are expressed at the NMJ at an age when synapse elimination is occurring in three different muscles. Several MHCI mRNA splice variants were detected in hippocampus, but not at the NMJ. Together, these results establish the first profile of MHCI gene expression at the developing NMJ, and demonstrate that MHCI gene expression is under tight spatial and temporal regulation in the nervous system. They also identify more than a dozen MHCIs that could play important roles in regulating synaptic transmission and plasticity in the central and peripheral nervous systems.

Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome.

Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection.

Which molecules regulate synaptic brain asymmetries?

Which molecules regulate synaptic brain asymmetries?

Neuronal major histocompatibility complex class I molecules are implicated in the generation of asymmetries in hippocampal circuitry

Left-right asymmetry is a fundamental feature of higher-order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluR2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of 2 subunits affects the properties of NMDARs and generates two populations of synapses, '2-dominant' and '2-non-dominant' synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of β2-microglobulin (β2m)-deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity-dependent refinement of neuronal connections and normal synaptic plasticity. We found that β2m proteins were localised in hippocampal synapses in wild-type mice. NMDA EPSCs in β2m-deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25-6981, an 2 subunit-selective antagonist, with those in '2-dominant' synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the β2m-deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of '2-dominant' synapses. These observations indicate that the β2m-deficient hippocampus lacks '2-non-dominant' synapses and circuit asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.

Structural mechanism of ER retrieval of MHC class I by cowpox.

Author SummaryViruses encode a wide array of proteins whose principle function is to disable the surveillance and effector functions of the immune system. A common viral target is the MHC class I antigen processing and presentation pathway, which is a potent mechanism used by the host for the detection and killing of virally infected cells. In this study we have delineated the immune evasion mechanism of the cowpox-encoded CPXV203 protein, which is known to potently block the normal trafficking of MHCI from the endoplasmic reticulum (ER) to the plasma membrane. CPXV203 does this by highjacking an ER-retrieval system that usually serves to capture defective, chaperone complexed MHCI proteins in the Golgi and send them to the ER. We show that CPXV203 adopts a compact beta-sandwich structure and engages evolutionarily conserved MHCI determinants that are located under the peptide-binding platform. The viral protein binds a variety of different MHCI proteins weakly at the pH found in the ER, but the affinity and half-life are significantly augmented at the more acidic conditions found in the Golgi. Together these data suggest that CPXV203 works cooperatively with the endogenous ER retrieval process to promiscuously target fully assembled MHCI, thereby preventing T-cell killing of cowpox infected cells.

MHC class I protein is expressed by neurons and neural progenitors in mid-gestation mouse brain

Proteins of the major histocompatibility complex class I (MHCI) are known for their role in the vertebrate adaptive immune response, and are required for normal postnatal brain development and plasticity. However, it remains unknown if MHCI proteins are present in the mammalian brain before birth. Here, we show that MHCI proteins are widely expressed in the developing mouse central nervous system at mid-gestation (E9.5-10.5). MHCI is strongly expressed in several regions of the prenatal brain, including the neuroepithelium and olfactory placode. MHCI is expressed by neural progenitors at these ages, as identified by co-expression in cells positive for neuron-specific class III β-tubulin (Tuj1) or for Pax6, a marker of neural progenitors in the dorsal neuroepithelium. MHCI is also co-expressed with nestin, a marker of neural stem/progenitor cells, in olfactory placode, but the co-localization is less extensive in other regions. MHCI is detected in the small population of post-mitotic neurons that are present at this early stage of brain development, as identified by co-expression in cells positive for neuronal microtubule-associated protein-2 (MAP2). Thus MHCI protein is expressed during the earliest stages of neuronal differentiation in the mammalian brain. MHCI expression in neurons and neural progenitors at mid-gestation, prior to the maturation of the adaptive immune system, is consistent with MHCI performing non-immune functions in prenatal brain development. These results raise the possibility that disruption of the levels and/or patterns of MHCI expression in the prenatal brain could contribute to the pathogenesis of neurodevelopmental disorders.

Microfluidic Devices Modulate Tumor Cell Line Susceptibility to NK Cell Recognition

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

Neuronal MHC Class I Molecules are Involved in Excitatory Synaptic Transmission at the Hippocampal Mossy Fiber Synapses of Marmoset Monkeys

Several recent studies suggested a role for neuronal major histocompatibility complex class I (MHCI) molecules in certain forms of synaptic plasticity in the hippocampus of rodents. Here, we report for the first time on the expression pattern and functional properties of MHCI molecules in the hippocampus of a nonhuman primate, the common marmoset monkey (Callithrix jacchus). We detected a presynaptic, mossy fiber-specific localization of MHCI proteins within the marmoset hippocampus. MHCI molecules were present in the large, VGlut1-positive, mossy fiber terminals, which provide input to CA3 pyramidal neurons. Furthermore, whole-cell recordings of CA3 pyramidal neurons in acute hippocampal slices of the common marmoset demonstrated that application of antibodies which specifically block MHCI proteins caused a significant decrease in the frequency, and a transient increase in the amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in CA3 pyramidal neurons. These findings add to previous studies on neuronal MHCI molecules by describing their expression and localization in the primate hippocampus and by implicating them in plasticity-related processes at the mossy fiber–CA3 synapses. In addition, our results suggest significant interspecies differences in the localization of neuronal MHCI molecules in the hippocampus of mice and marmosets, as well as in their potential function in these species.Electronic supplementary materialThe online version of this article (doi:10.1007/s10571-010-9510-3) contains supplementary material, which is available to authorized users.

Layers upon Layers: MHC Class I Acts in the Retina to Influence Thalamic Segregation

Recent research has introduced the major histocompatibility complex class I (MHCI) genes as unexpected players in structural and synaptic plasticity in the central nervous system. In this issue of Neuron, Xu et al. redirect current theory by providing strong evidence for the inner retina as a site of action of MHCI proteins in retinogeniculate refinement.

Species-Specific Effects of HIV-1 Nef-Mediated MHC-I Downmodulation

In vitro studies have revealed that human immunodeficiency virus-1 (HIV-1) Nef functionally interacts with amino acid residues in the cytoplasmic tail of major histocompatibility complex class I (MHC-I) molecules, reducing their expression on the cell surface and protecting them from cytotoxic T lymphocyte (CTL) lysis. To obtain a better understanding of Nef's effects in vivo, it would be helpful to have a mouse model system. However, it is not known whether Nef will affect murine MHC-I proteins. We find that Nef downmodulates human MHC-I HLA-A2 more efficiently than murine MHC-I molecules in HeLa cells and that Nef does not function efficiently in murine endothelial cells. Studies with chimeric molecules indicate that the MHC-I cytoplasmic tail is primarily responsible for species-specific differences. However, there are also effects attributable to the extracellular domain.