Physical and functional association of the major histocompatibility complex class I heavy chain alpha3 domain with the transporter associated with antigen processing.

CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits.

LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

Transport protein genes in the murine MHC: possible implications for antigen processing.

T lymphocyte activation requires recognition by the T cell of peptide fragments of foreign antigen bound to a self major histocompatibility complex (MHC) molecule. Genetic evidence suggests that part of the class II region of the MHC influences the expression, in trans, of MHC class I antigens on the cell surface, by regulating the availability of peptides that bind to and stabilize the class I molecule. Two closely related genes in this region, HAM1 and HAM2, were cloned and had sequence similarities to a superfamily of genes involved in the ATP-dependent transport of a variety of substrates across cell membranes. Thus, these MHC-linked transport protein genes may be involved in transporting antigen, or peptide fragments thereof, from the cytoplasm into a membrane-bounded compartment containing newly synthesized MHC molecules.

Structure and function of genes in the MHC class II region.

Genes required for antigen processing map to the MHC class II region. For the endogenous (class I) antigen processing pathway, many hypotheses concerning the structure and function of the corresponding gene products have been verified during the past year. The identity of the gene(s) involved in the exogenous (class II) pathway remains to be determined.

Genes in the MHC that may affect antigen processing.

Recent evidence indicates that genes required for antigen processing are present in the class II region of the MHC. Two new classes of MHC genes that may encode much of the machinery required in the class I (endogenous) antigen-processing pathway have been discovered. There is evidence that genes required in the class II pathway also reside in this region.

Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta-cell lines.

The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.

Pathways for the processing and presentation of antigens to T cells.

Two pathways exist within vertebrate cells to generate peptides for recognition by T cells. The "endogenous" pathway provides peptides to MHC class I molecules for presentation to CD8+ T cells. These peptides are derived from proteins synthesized or residing in the cytoplasm or nucleus, and involves proteasomes and the ubiquitin pathway of protein degradation, as well as a specific peptide transporter (TAP) that allows these peptides access to the lumen of the endoplasmic reticulum. The exogenous pathway provides peptides to MHC class II molecules for presentation to CD4+ T cells. These peptides are derived from extracellular antigens taken up by endocytosis and degraded in the endosomal/lysosomal pathway. Peptide loading of MHC class II molecules requires the presence of a molecule (H-2M in mouse, HLA-DM in humans) that is structurally related to MHC class II molecules, but the mechanistic basis of this requirement is unknown. The class II region of the MHC contains a cluster of genes encoding proteins involved in antigen processing, including genes for two proteasome subunits (LMP2 and LMP7), the peptide transporter heterodimer (TAP1 and TAP2), and the H-2M/HLA-DM molecule (Ma and Mb, or DMA and DMB).

Post-translational processing of a major histocompatibility complex-encoded proteasome subunit, LMP-2.

Proteasomes are abundant, multisubunit protein complexes found in the cytoplasm and nucleus of eukaryotic cells that catalyze both ubiquitin-dependent and ubiquitin-independent protein degradation. In addition to their role in normal protein turnover, proteasomes are believed to be involved in the production of most antigenic peptides presented to T cells by major histocompatibility complex (MHC) class I molecules. A distinct subset of mouse proteasomes contain a subunit called LMP-2, which is encoded within the MHC. Here we demonstrate that a previously isolated proteasome cDNA clone encodes the LMP-2 subunit, and that two distinct forms of this subunit may be found in the proteasome complex. One form probably corresponds to the primary translation product, whereas the second form appears to be post-translationally processed by removal of the amino-terminal 20 amino acids. Determination of the location of intron/exon boundaries in the Lmp-2 gene indicated that these residues correspond precisely to the first exon of the gene.

Genomic organization of the mouse Lmp-2 gene and characteristic structure of its promoter.

Major histocompatibility complex (MHC) class-I molecules present antigenic peptide fragments to cytotoxic T-cells. The peptides are generated in the course of antigen processing from endogenously synthesized cytosolic proteins, and transported into the endoplasmic reticulum to associate with an MHC class-I molecule. So far, at least four genes, Lmp-2, -7, and Tap-1, -2, have been identified between the Pb and Ob genes of the mouse MHC class-II region. The genomic organization of mouse Lmp-2, a gene encoding a subunit of a large intracellular protein complex, was studied. A genomic clone has been isolated that covers the entire mouse Lmp-2 gene. We have determined the nucleotide sequence of the region encompassing the whole Lmp-2 gene and three exons of Tap-1, which spans 8 kb in the mouse genome. The two genes are situated in opposite directions. The transcription start points (tsp) of the two genes, identified by primer extension analysis, are only 118 bp apart. Both promoter regions upstream from the tsp have neither TATA consensus sequences nor other regulatory elements, like an interferon-response element, in spite of their interferon-inducible expression. The Lmp-2 sequence from a non-obese diabetic (NOD) mouse, a model animal for autoimmune diabetes, was compared with that from a Balb/c mouse.

Cloning and characterization of mouse Lmp3 cDNA, encoding a proteasome beta subunit.

We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.

Major histocompatibility complex-linked transport proteins and antigen processing.

The major histocompatibility complexes of mice, rats and humans each contain a pair of related genes, Tap-1 and Tap-2, that encode members of a large superfamily of proteins having similar structure and function. The TAP-1 (previously called HAM1 in the mouse) and TAP-2 (HAM2) proteins each contain 6-8 predicted membrane-spanning alpha helices, and a cytoplasmic domain containing a putative ATP-binding site. Recent evidence suggests that a functional TAP-1/TAP-2 heterodimer is required for efficient presentation of antigens to CD8+ cytotoxic T cells. This heterodimer resides in the membrane of the endoplasmic reticulum (ER), and probably functions to transport peptides (produced in the cytoplasm) into the ER lumen for binding to MHC class I molecules.

The LMP antigens: a stable MHC-controlled multisubunit protein complex.

In addition to encoding the well-known class I (H-2), II (Ia), and III (complement components C2, C4, and factor B) antigens, the murine MHC controls the expression of a large, intracellular protein complex of unknown function. This complex is composed of a large number of noncovalently linked low molecular weight polypeptide subunits (hence the name, LMP) which are biochemically, serologically, and genetically distinct from class I, II, and III antigens. Only two of these subunits display electrophoretic polymorphism within the standard inbred mouse strains, and both of these polymorphisms map within the H-2 complex, between the H-2K and I-A subregions. The remainder of the LMP complex subunits have not been mapped, and may be encoded elsewhere in the genome. A biochemically similar complex has been detected in human cells, although linkage to HLA remains to be established. In this article we will review the biochemistry, serology, and genetics of the LMP antigens, and will speculate on their biological function.

A molecular model of MHC class-I-restricted antigen processing.

Cells of higher vertebrates have evolved mechanisms that allow a sample of their intracellular contents to be available for surveillance by the immune system. This display of intracellular material is in the form of peptides bound to cell surface major histocompatibility complex (MHC) class I molecules. In this review, John Monaco presents a model of the mechanisms by which this takes place, based on the recent identification of a number of new genes in the MHC.

Identification of a fourth class of proteins linked to the murine major histocompatibility complex.

A series of proteins biochemically and genetically distinct from previously defined murine major histocompatibility complex class I and class II antigens is precipitated by a congeneic anti-H-2d antiserum. Sixteen such proteins have been defined, exhibiting a range of molecular weights (approximately 15,000-30,000) and isoelectric points (pI approximately 4-9). These proteins are not glycosylated, and they are probably not expressed at the cell surface. They are expressed most strongly in normal macrophages and macrophage cell lines and are also found in fibroblasts, B, T, and null cell lines. The genes controlling the expression of these proteins have been tentatively mapped within the H-2 complex, between the K and I-A subregions. Three alleles have been defined: mice of the H-2 haplotypes b and q possess a "null" allele, i.e., do not express any demonstrable protein product. Mice of the d haplotype cane be distinguished by their two-dimensional gel pattern from mice of all other positive H-2 types tested thus far (a, k, f, s, and ja).

Characterization and mapping of the gene encoding mouse proteasome subunit DELTA (Lmp19).

The proteasome subunit DELTA is unusually closely related to the major histocompatibility complex (MHC)-linked proteasome subunit, LMP2. The sequence of a mouse cDNA for DELTA confirms that this 22,100 M(r) proteasome subunit is highly conserved across species. Sequence analysis of the mouse gene encoding DELTA, designated Lmp19, indicates that it consists of six exons and five introns, similar to the Lmp2 gene. The 5' upstream region lacks a TATA regulatory sequence, which is also absent from proteasome genes isolated from Drosophila. BXD recombinant inbred (RI) mice were used to map the potential chromosomal location of Lmp19, and revealed that the DELTA subunit has related sequences present on two different mouse chromosomes, chromosomes 1 and 11. Typing of 89 progeny from a C57BL/6J X Mus spretus DNA backcross panel (BSS) confirmed the chromosome 1 assignment. Southern hybridization with a polymerase chain reaction-generated Lmp19 intron 2-specific probe indicates that the Lmp19 genomic clone corresponds to the sequence on chromosome 11, and further suggests that the chromosome 1 copy represents a processed pseudogene (Lmp19-ps1).