Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain

We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.     

Newly discovered viral E3 ligase pK3 induces endoplasmic reticulum-associated degradation of class I major histocompatibility proteins and their membrane-bound chaperones

Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.     

The viral E3 ubiquitin ligase mK3 uses the Derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class I proteins

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.     

Requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class I proteins by the viral immune evasion molecule mK3

Recent studies suggest that certain viral proteins co-opt endoplasmic reticulum (ER) degradation pathways to prevent the surface display of major histocompatibility complex class I molecules to the immune system. A novel example of such a molecule is the mK3 protein of gammaherpesvirus 68. mK3 belongs to an extensive family of structurally similar viral and cellular proteins that function as ubiquitin ligases using a conserved RING-CH domain. In the specific case of mK3, it selectively targets the rapid degradation of nascent class I heavy chains in the ER while they are associated with the class I peptide-loading complex (PLC). We present here evidence that the PLC imposes a relative proximity and/or orientation on the RING-CH domain of mK3 that is required for it to specifically target class I molecules for degradation. Furthermore, we demonstrate that full assembly of class I molecules with peptide is not a prerequisite for mK3-mediated degradation. Surprisingly, although the cytosolic tail of class I is required for rapid mK3-mediated degradation, we observed that a class I mutant lacking lysine residues in its cytosolic tail was ubiquitinated and degraded in the presence of mK3 in a manner indistinguishable from wild-type class I molecules. These findings are consistent with a "partial dislocation" model for turnover of ER proteins and define some common features of ER degradation pathways initiated by structurally distinct herpesvirus proteins.     

Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

The major histocompatibility complex (MHC) class I antigens contain a light chain β₂-microglobulin, non-covalently associated to the transmembrane heavy α-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with β₂-microglobulin, and we recently found that activated C1s complement was capable of cleaving β₂-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22 000 and 24 000 g/ mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the α₂-andα₃-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-A^d). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the α₁andα₂-domains which represent the binding site for atnigenic peptide.     

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.     

The human cytomegalovirus US8 glycoprotein binds to major histocompatibility complex class I products

Human cytomegalovirus US8 is a type I membrane protein that partially colocalizes with cellular endosomal and lysosomal proteins. Although US8 does not have discernible effects on the processing and cell surface distribution of major histocompatibility complex (MHC) class I products, we have demonstrated that US8 binds to MHC class I heavy chains in the endoplasmic reticulum.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.     

The human cytomegalovirus US10 gene product delays trafficking of major histocompatibility complex class I molecules

Human cytomegalovirus (HCMV) US10 encodes a glycoprotein that binds to major histocompatibility complex (MHC) class I heavy chains. While expression of US10 delays the normal trafficking of MHC class I molecules out of the endoplasmic reticulum, US10 does not obviously facilitate or inhibit the action of two other HCMV-encoded MHC class I binding proteins, US2 and US11.     

Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Free major histocompatibility complex class I heavy chain is preferentially targeted for degradation by human T-cell leukemia/lymphotropic virus type 1 p12(I) protein

Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.     

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.     

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP

ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.     

Peptide-receptive major histocompatibility complex class I molecules cycle between endoplasmic reticulum and cis-Golgi in wild-type lymphocytes

Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ER-Golgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptide-receptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.     

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.