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.     

Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly

The assembly and peptide loading of major histocompatibility complex Class I molecules within the endoplasmic reticulum are essential for antigen presentation at the cell surface and are facilitated by the peptide-loading complex. The formation of a mixed disulfide between the heavy chain of Class I and components of the loading complex (ERp57, protein disulfide isomerase, and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is coincident with the stabilization of the alpha2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57, and tapasin was observed and shown to be stabilized by a disulfide between both tapasinheavy chain and tapasin-ERp57. No disulfides were observed between ERp57 and heavy chain within the loading complex. The results provide a detailed evaluation of the various transient disulfides formed within the peptide-loading complex during biosynthesis. In addition, the absence of the disulfide between tapasin and heavy chain in TAP-deficient cells indicates that a change in the spatial organization of tapasin and heavy chain occurs following assembly into the loading complex.     

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.     

Molecular architecture of the TAP-associated MHC class I peptide-loading complex

Tapasin organizes the peptide-loading complex (PLC) by recruiting peptide-receptive MHC class I (MHC-I) and accessory chaperones to the N-terminal regions of the TAP subunits TAP1 and TAP2. Despite numerous studies have shown that the formation of the PLC is essential to facilitate proper MHC-I loading, the molecular architecture of this complex is still highly controversial. We studied the stoichiometry of the PLC by blue native-PAGE in combination with Ab-shift assays and found that TAP/tapasin complexes exist at steady state as a mixture of two distinct oligomers of 350 and 450 kDa. Only the higher m.w. complex contains MHC-I and disulfide-linked tapasin/ER60 conjugates. Moreover, we show for the first time to our knowledge that the fully assembled PLC comprises two tapasin, two ER60, but only one complex of MHC-I and calreticulin. Based hereon we postulate that the TAP subunits alternate in the recruitment and loading of a single MHC-I.     

Physical association of the K3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class I molecules with impaired peptide and beta(2)-microglobulin assembly

To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.     

Peptide selection by class I molecules of the major histocompatibility complex

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in providing peptides with the correct residues at the carboxyl terminus.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

A nonpolymorphic class I gene in the murine major histocompatibility complex

DNA sequence analysis of a class I gene (Q10), which maps to the Qa2,3 locus in the C57BL/10 (H-2b haplotype) mouse, reveals that it is almost identical to a cDNA clone (pH16) isolated from a SWR/J (H-2q haplotype) mouse liver cDNA library. Exon 5, in particular, has an unusual structure such that a polypeptide product is unlikely to be anchored in the cell membrane. Our findings suggest that the two sequences are derived from allelic class I genes, which are nonpolymorphic, in contrast to H-2K allelic sequences from the same mice, and they may encode liver-specific polypeptides of unknown function. Our previous studies indicate that the Q10 gene is a potential donor gene for the generation of mutations at the H-2K locus by inter-gene transfer of genetic information. Thus the lack of polymorphism in class I genes at the Q10 locus implies either that they are not recipients for such exchanges or that selective pressure prevents the accumulation of mutations in genes at this locus.     

Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.     

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.     

Mapping of class I DNA sequences within the human major histocompatibility complex

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the beta 2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to beta 2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed beta 2m-specific mAb B1G6 does not recognize the beta 2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine beta 2m which is strongly influenced by the nature of the heavy chain with which the beta 2m is associated.     

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d)

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Summary. Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the β2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to β2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed β2m-specific mAb B1G6 does not recognize the β2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine β2m which is strongly influenced by the nature of the heavy chain with which the β2m is associated.     

Direct interaction of major histocompatibility complex class II-derived peptides with class Ia phosphoinositide 3-kinase results in dose-dependent stimulatory effects

Peptides corresponding to residues 65-79 of human lymphocyte antigen class II sequence (DQA*03011) are cell-permeable and at high concentrations block activation of protein kinase B/Akt and p70-S6 kinase in T-cells, effects attributed to inhibition of phosphoinositide (PI) 3-kinase activity. To understand the molecular basis of this, we analyzed the effect this peptide had on activity of class I PI 3-kinases. Although there was no effect on the activity of class Ib PI 3-kinase or on the protein kinase activity of class I PI 3-kinases, there was a biphasic effect on lipid kinase activity of the class Ia enzymes. There was an inhibition of activity at higher peptide concentrations because of a formation of insoluble complexes between peptide and enzyme. Conversely, at lower peptide concentrations there was a profound activation of PI 3-kinase activity of class Ia PI 3-kinases. Studies of peptide variants revealed that all active peptides conform to heptad repeat motifs characteristic of coiled-coil helices. Surface plasmon resonance studies confirmed direct sequence-specific binding of active peptide to the p85alpha adapter subunit of class Ia PI 3-kinase. Active peptides also activated protein kinase B and extracellular signal-regulated kinase (ERK) in vivo in a wortmannin-sensitive manner while reducing recoverable cellular p85 levels. These results indicate that the human lymphocyte antigen class II-derived peptides regulate PI 3-kinase by direct interaction, probably via the coiled-coil domain. These peptides define a novel mechanism of regulating PI 3-kinase and will provide a useful tool for specifically dissecting the function of class Ia PI 3-kinase in cells and for probing structure-function relationships in the class Ia PI 3-kinase heterodimers.