Structural and serological similarity of MHC-linked LMP and proteasome (multicatalytic proteinase) complexes

Major histocompatibility complex (MHC) class I molecules associate with peptides derived from endogenously synthesized antigens. Cytotoxic T-lymphocytes can thus scan class I molecules and bound peptide on the surface of cells for foreign antigenic determinants. Recent evidence demonstrates that the products of trans-acting, non-class I genes in the class II region of the MHC are required in the class I antigen-processing pathway. There are genes (called HAM1 and HAM2 in the mouse) in this region that encode proteins postulated to be involved in the transport of peptide fragments into the endoplasmic reticulum for association with newly synthesized class I molecules. But, the mechanism by which such peptide fragments are produced remains a mystery. At least two genes encoding subunits of the low-molecular mass polypeptide (LMP) complex are tightly linked to the HAM1 and HAM2 genes. We show that the LMP complex is closely related to the proteasome (multicatalytic proteinase complex), an intracellular protein complex that has multiple proteolytic activities. We speculate that the LMP complex may have a role in MHC class I antigen processing, and therefore that the MHC contains a cluster of genes required for distinct functions in the antigen processing pathway.     

ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum

The ability of killer T cells carrying the CD8 antigen to detect tumours or intracellular pathogens requires an extensive display of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of potential target cells. These peptides are derived from almost all intracellular proteins and reveal the presence of foreign pathogens and mutations. How cells produce thousands of distinct peptides cleaved to the precise lengths required for binding different MHC class I molecules remains unknown. The peptides are cleaved from endogenously synthesized proteins by the proteasome in the cytoplasm and then trimmed by an unknown aminopeptidase in the endoplasmic reticulum (ER). Here we identify ERAAP, the aminopeptidase associated with antigen processing in the ER. ERAAP has a broad substrate specificity, and its expression is strongly upregulated by interferon-gamma. Reducing the expression of ERAAP through RNA interference prevents the trimming of peptides for MHC class I molecules in the ER and greatly reduces the expression of MHC class I molecules on the cell surface. Thus, ERAAP is the missing link between the products of cytosolic processing and the final peptides presented by MHC class I molecules on the cell surface.     

Second proteasome-related gene in the human MHC class II region

Antgen processing involves the generation of peptides from cytosolic proteins and their transport into the endoplasmic reticulum where they associate with major histocompatibility complex (MHC) class I molecules. Two genes have been identified in the MHC class II region, RING4 and RING11 in humans, which are believed to encode the peptide transport proteins. Attention is now focused on how the transporters are provided with peptides. The proteasome, a large complex of subunits with multiple proteolytic activities, is a candidate for this function. Recently we reported a proteasome-related sequence, RING10, mapping between the transporter genes. Here we describe a second human proteasome-like gene, RING12, immediately centromeric of the RING4 locus. Therefore RING12, 4, 10 and 11 form a tightly linked cluster of interferon-inducible genes within the MHC with an essential role in antigen processing.     

The major substrates for TAP in vivo are derived from newly synthesized proteins

The transporter associated with antigen processing (TAP) is a member of the family of ABC transporters that translocate a large variety of substrates across membranes. TAP transports peptides from the cytosol into the endoplasmic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system. Here we follow the lateral mobility of TAP in living cells. TAP's mobility increases when it is inactive and decreases when it translocates peptides. Because TAP activity is dependent on substrate, the mobility of TAP is used to monitor the intracellular peptide content in vivo. Comparison of the diffusion rates in peptide-free and peptide-saturated cells indicates that normally about one-third of all TAP molecules actively translocate peptides. However, during an acute influenza infection TAP becomes fully employed owing to the production and degradation of viral proteins. Furthermore, TAP activity depends on continuing protein translation. This implies that MHC class I molecules mainly sample peptides that originate from newly synthesized proteins, to ensure rapid presentation to the immune system.     

Homology of proteasome subunits to a major histocompatibility complex-linked LMP gene.

The class II region of the major histocompatibility complex (MHC) contains genes encoding at least two subunits of a large, intracellular protein complex (the low molecular mass polypeptide, or LMP, complex). This complex is biochemically similar to the proteasome, an abundant and well conserved protein complex having multiple proteolytic activities. Here we report the isolation of a complementary DNA corresponding to one of the subunits of the LMP complex, LMP-2. The protein predicted from this cDNA sequence closely matches the amino-terminal peptide sequence of a rat proteasome subunit, confirming that the proteasome and the LMP complex share polypeptide subunits. The LMP-2 gene is tightly linked to HAM1, a gene thought to be required for translocating peptide fragments of endogenous antigens into the endoplasmic reticulum for association with MHC class I molecules. These observations suggest that the LMP complex may be responsible for generating peptides from cytoplasmic antigen during antigen processing.     

ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells

Induction of cytotoxic T-cell immunity requires the phagocytosis of pathogens, virus-infected or dead tumour cells by dendritic cells. Peptides derived from phagocytosed antigens are then presented to CD8+ T lymphocytes on major histocompatibility complex (MHC) class I molecules, a process called "cross-presentation". After phagocytosis, antigens are exported into the cytosol and degraded by the proteasome. The resulting peptides are thought to be translocated into the lumen of the endoplasmic reticulum (ER) by specific transporters associated with antigen presentation (TAP), and loaded onto MHC class I molecules by a complex "loading machinery" (which includes tapasin, calreticulin and Erp57). Here we show that soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, peptides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I molecules. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER-phagosome mix compartment.     

Immune recognition of a human renal cancer antigen through post-translational protein splicing

Cytotoxic T lymphocytes (CTLs) detect and destroy cells displaying class I molecules of the major histocompatibility complex (MHC) that present oligopeptides derived from aberrant self or foreign proteins. Most class I peptide ligands are created from proteins that are degraded by proteasomes and transported, by the transporter associated with antigen processing, from the cytosol into the endoplasmic reticulum, where peptides bind MHC class I molecules and are conveyed to the cell surface. C2 CTLs, cloned from human CTLs infiltrating a renal cell carcinoma, kill cancer cells overexpressing fibroblast growth factor-5 (FGF-5). Here we show that C2 cells recognize human leukocyte antigen-A3 MHC class I molecules presenting a nine-residue FGF-5 peptide generated by protein splicing. This process, previously described strictly in plants and unicellular organisms, entails post-translational excision of a polypeptide segment followed by ligation of the newly liberated carboxy-terminal and amino-terminal residues. The occurrence of protein splicing in vertebrates has important implications for the complexity of the vertebrate proteome and for the immune recognition of self and foreign peptides.     

Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters

Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.     

Sequence analysis of peptides bound to MHC class II molecules.

CD4 T cells recognize peptide fragments of foreign proteins bound to self class II molecules of the major histocompatibility complex (MHC). Naturally processed peptide fragments bound to MHC class II molecules are peptides of 13-17 amino acids which appear to be precessively truncated from the carboxy terminus, perhaps after binding to the MHC class II molecule. The finding of predominant self peptides has interesting implications for antigen processing and self-non-self discrimination.     

Empty MHC class I molecules come out in the cold

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.     

Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation.

Major histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette encoded by the MHC class II region seem to be responsible for this transport. Genes coding for two subunits of the '20S' proteasome (a multicatalytic proteinase) have been found in the vicinity of the two transporter genes in the MHC class II region, indicating that the proteasome could be the unknown proteolytic entity in the cytosol involved in the generation of MHC class I-binding peptides. By introducing rat genes encoding the MHC-linked transporters into a human cell line lacking both transporter and proteasome subunit genes, we show here that the MHC-encoded proteasome subunit are not essential for stable MHC class I surface expression, or for processing and presentation of antigenic peptides from influenza virus and an intracellular protein.     

Physical association between MHC class I molecules and immunogenic peptides

Antigenic peptides are presented to T lymphocytes by major histocompatibility complex (MHC) molecules. The binding of peptides to MHC class II molecules has been demonstrated directly, and is found to correlate with the ability of specific class II alleles to restrict the T-cell response to specific peptides. By comparison, a direct demonstration of a physical association between antigenic peptides and MHC class I molecules has proved difficult. A recent report shows that it is possible, however, and the three-dimensional structure of a class I MHC molecule illustrates the site where such binding must occur. Here we describe a simple assay which measures the binding of radiolabelled MHC class I molecules to peptides bound to a solid phase support. We find that class I molecules bind specifically to peptides known to be antigenic for class I-restricted cytotoxic T lymphocytes. Peptides which are recognized by cytotoxic T lymphocytes bind not only to the restricting MHC class I molecule but also to other class I molecules. Our results suggest that quantitative differences in the peptide/MHC class I interaction may influence the-pattern of MHC restriction observed in vivo.     

MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters

The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system. Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER). In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism. Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC. Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance. Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway. Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.     

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.     

A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC

It is now possible to paint a detailed picture of how cytoplasmic proteins are handled by the immune system. They are apparently degraded in the cytoplasm into peptides. These are then transported into the endoplasmic reticulum where they encounter class I major histocompatibility complex (MHC) molecules. Once loaded with peptide, the HLA molecules move through the Golgi apparatus to the cell membrane. Until recently, it had not been established how peptides without signal sequences cross the ER membrane. However, a number of papers have now described a pair of membrane transporter genes of the ABC (ATP-binding cassette) super-family which are attractive candidates for this function. Both transporter genes, which may encode two halves of a heterodimer, are situated in the class II region of the MHC. There is evidence that other putative components of the processing machinery, the LMPs (low molecular mass polypeptides), are also encoded in the MHC. Similarities between the properties of the LMPs and a large intracellular protease complex, called proteasome, have led to the suggestion that LMPs are involved in processing antigens. We have now identified a human gene with sequence homology to proteasome components. Remarkably, this gene maps between the two putative peptide transporter genes.     

Specific binding of antigenic peptides to cell-associated MHC class I molecules

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.     

Rapid degradation of a large fraction of newly synthesized proteins by proteasomes

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.     

Cellular peptide composition governed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present peptides derived from cellular proteins to cytotoxic T lymphocytes (CTLs), which check these peptides for abnormal features. How such peptides arise in the cell is not known. Here we show that the MHC molecules themselves are substantially involved in determining which peptides occur intracellularly: normal mouse spleen cells identical at all genes but MHC class I express different patterns of peptides derived from cellular non-MHC proteins. We suggest several models to explain this influence of MHC class I molecules on cellular peptide composition.     

抗原处理相关转运体( TAP) 的结构和功能研究进展

摘要: 抗原处理相关转运体( TAP) 属于 ABC( ATP-binding cassette) 转运体超家族的 B 亚家族,是由主要组织相容性 复合体( Major histocompatibility complex ,MHC) 上 2 个紧密连锁的基因 TAP1 和 TAP2 编码蛋白组成的异二聚体,位 于内质网和顺式高尔基膜上,功能是将抗原肽从细胞质转运进入内质网池,组装到 MHC I 类分子上构成复合体。 TAP 蛋白的缺乏和突变会影响抗原肽的提呈,最终损害免疫应答功能。     

Processing of a minimal antigenic peptide alters its interaction with MHC molecules

Before their recognition by T lymphocytes, protein antigens generally require processing by antigen-presenting cells. In a poorly understood series of events, the protein antigen is internalized, transformed and re-expressed on the surface of the antigen-presenting cell in association with gene products of the major histocompatibility complex (MHC). Small peptides derived from the native protein can be recognized in the absence of antigen processing, suggesting that processing involves proteolytic degradation. These peptides are thought to mimic the naturally produced peptide fragment. We describe here a synthetic peptide antigen of this type which does not require processing but which is nevertheless further processed by splenic antigen-presenting cells. Interestingly, this processing event specifically alters the interaction of the peptide with the class II MHC (Ia) molecule, markedly affecting both its potency as an antigen in vitro and its immunogenicity in vivo (IR gene control).