Assembly and Function of the Major Histocompatibility Complex (MHC) I Peptide-loading Complex Are Conserved Across Higher Vertebrates

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.     

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2⁺ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2⁺ I-A^k conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2⁺ versus Ia.2⁻ I-A^k class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.     

A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins

MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.     

Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.     

MICA/B蛋白在不同宫颈病变组织中的表达及意义

目的:探讨主要组织相容性复合物I类相关蛋白A/B(MICA/B)在不同宫颈病变组织及宫颈细胞系中的表达及定位。方法:采用免疫组织化学SP法检测宫颈炎症组织、高级别鳞状上皮内病变(high-grade squamous intraepithelial lesions, HSIL)及宫颈鳞癌(cervical squamous cell carcinoma, CSCC)组织中MICA/B蛋白的表达情况。采用免疫荧光化学与激光共聚焦显微术结合的方法研究3种宫颈癌细胞系C33a(HPV-)、Siha(HPV16+)、Hela(HPV18+)及正常宫颈上皮细胞系H8中MICA/B的表达和定位。结果:MICA/B蛋白主要表达定位于细胞浆,部分细胞核,在宫颈鳞癌组织中阳性表达率(83.3%、81.8%)高于宫颈炎症组织(39.3%、44.0%),差异具有统计学意义(均有P0.001);MICA蛋白在HSIL组织的阳性表达率(81.8%)高于宫颈炎症组织(39.3%),差异具有统计学意义(P=0.002);与分化程度、临床分期、淋巴结转移等临床病理参数之间比较无统计学差异(P0.05)。结论:MICA蛋白随着宫颈组织病变的加重阳性表达率逐渐增高,MICB蛋白在宫颈癌组织的表达高于宫颈炎症组织。提示MICA/B蛋白可为宫颈癌的诊断及靶向治疗提供新方向。     

Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex

In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8⁺ T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition.     

The Same Major Histocompatibility Complex Polymorphism Involved in Control of HIV Influences Peptide Binding in the Mouse H-2Ld System

Single-site polymorphisms in human class I major histocompatibility complex (MHC) products (HLA-B) have recently been shown to correlate with HIV disease progression or control. An identical single-site polymorphism (at residue 97) in the mouse class I product H-2L^d influences stability of the complex. To gain insight into the human polymorphisms, here we examined peptide binding, stability, and structures of the corresponding L^d polymorphisms, Trp⁹⁷ and Arg⁹⁷. Expression of L^dW97 and L^dR97 genes in a cell line that is antigen-processing competent showed that L^dR97 was expressed at higher levels than L^dW97, consistent with enhanced stability of self-peptide·L^dR97 complexes. To further examine peptide-binding capacities of these two allelic variants, we used a high affinity pep-L^d specific probe to quantitatively examine a collection of self- and foreign peptides that bind to L^d. L^dR97 bound more effectively than L^dW97 to most peptides, although L^dW97 bound more effectively to two peptides. The results support the view that many self-peptides in the L^d system (or the HLA-B system) would exhibit enhanced binding to Arg⁹⁷ alleles compared with Trp⁹⁷ alleles. Accordingly, the self-peptide·MHC-Arg⁹⁷ complexes would influence T-cell selection behavior, impacting the T-cell repertoire of these individuals, and could also impact peripheral T cell activity through effects of self-peptide·L^d interacting with TCR and/or CD8. The structures of several peptide·L^dR97 and peptide·L^dW97 complexes provided a framework of how this single polymorphism could impact peptide binding.     

A Viral,Transporter Associated with Antigen Processing (TAP)-independent,High Affinity Ligand with Alternative Interactions Endogenously Presented by the Nonclassical Human Leukocyte Antigen E Class I Molecule

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8⁺ lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.     

Mechanisms of Function of Tapasin,a Critical Major Histocompatibility Complex Class I Assembly Factor

For their efficient assembly in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class I molecules require the specific assembly factors transporter associated with antigen processing (TAP) and tapasin, as well as generic ER folding factors, including the oxidoreductases ERp57 and protein disulfide isomerase (PDI), and the chaperone calreticulin. TAP transports peptides from the cytosol into the ER. Tapasin promotes the assembly of MHC class I molecules with peptides. The formation of disulfide‐linked conjugates of tapasin with ERp57 is suggested to be crucial for tapasin function. Important functional roles are also suggested for the tapasin transmembrane and cytoplasmic domains, sites of tapasin interaction with TAP. We show that interactions of tapasin with both TAP and ERp57 are correlated with strong MHC class I recruitment and assembly enhancement. The presence of the transmembrane/cytosolic regions of tapasin is critical for efficient tapasin–MHC class I binding in interferon‐γ‐treated cells, and contributes to an ERp57‐independent mode of MHC class I assembly enhancement. A second ERp57‐dependent mode of tapasin function correlates with enhanced MHC class I binding to tapasin and calreticulin. We also show that PDI binds to TAP in a tapasin‐independent manner, but forms disulfide‐linked conjugates with soluble tapasin. Thus, full‐length tapasin is important for enhancing recruitment of MHC class I molecules and increasing specificity of tapasin–ERp57 conjugation. Furthermore, tapasin or the TAP/tapasin complex has an intrinsic ability to recruit MHC class I molecules and promote assembly, but also uses generic folding factors to enhance MHC class I recruitment and assembly.     

Disruption of Multivesicular Body Vesicles Does Not Affect Major Histocompatibility Complex (MHC) Class II-Peptide Complex Formation and Antigen Presentation by Dendritic Cells

The antigen processing compartments in antigen-presenting cells (APCs) have well known characteristics of multivesicular bodies (MVBs). However, the importance of MVB integrity to APC function remains unknown. In this study, we have altered the ultrastructure of the MVB by perturbing cholesterol content genetically through the use of a deletion of the lipid transporter Niemann-Pick type C1 (NPC1). Immunofluorescence and electron microscopic analyses reveal that the antigen processing compartments in NPC1^−/− dendritic cells (DCs) have an abnormal ultrastructure in that the organelles are enlarged and the intraluminal vesicles are almost completely absent and those remaining are completely disorganized. MHC-II is restricted to the limiting membrane of these enlarged MVBs where it colocalizes with the peptide editor H2-DM. Curiously, proteolytic removal of the chaperone protein Invariant chain from MHC-II, degradation of internalized foreign antigens, and antigenic-peptide binding to nascent MHC-II are normal in NPC1^−/− DCs. Antigen-pulsed NPC1^−/− DCs are able to effectively activate antigen-specific CD4 T cells in vitro, and immunization of NPC1^−/− mice reveals surprisingly normal CD4 T cell activation in vivo. Our data thus reveal that the localization of MHC-II on the intraluminal vesicles of multivesicular antigen processing compartments is not required for efficient antigen presentation by DCs.     

Dynamics of Major Histocompatibility Complex Class I Association with the Human Peptide-loading Complex

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.     

Structural Requirements for Recognition of Major Histocompatibility Complex Class II by Membrane-associated RING-CH (MARCH) Protein E3 Ligases

MARCH E3 ligases play a key role in controlling MHC class II surface expression by regulated ubiquitination of a lysine residue in the β-chain. Little is known concerning how these enzymes target their specific substrates. Here we show that recognition of HLA-DR by MARCH proteins is complex. Several features associated with the transmembrane domain and bordering regions influence the overall efficiency of receptor internalization. A cluster of residues at the interface of the lipid bilayer and the cytosol plays the most important role in MARCH8 recognition of HLA-DRβ. Variation in this sequence also determines specificity of MARCH9 for HLA-DQ. Residues located in helical face four of HLA-DRβ together with a charged residue at the boundary with the stalk region also contribute significantly to recognition. Truncation analysis suggested that a dileucine-like motif in the DRβ cytoplasmic tail influences the efficiency of co-localization of HLA-DR with MARCH8. The DRβ-encoded acceptor lysine functioned optimally when placed in its natural location relative to the bilayer. In the DRα/DRβ dimer most other amino acids in the cytoplasmic tail could be substituted for alanine with minimal influence on function. Our data support a model whereby multiple features of HLA-DR are involved in substrate recognition by MARCH8. The single most important region is located at the interface between the transmembrane domain and the cytosol. Variation in sequence in this location between different class II isotypes controls efficiency of recognition by different MARCH E3 ligases.     

Characterization of Natural Human Nucleotide-binding Oligomerization Domain Protein 1 (Nod1) Ligands from Bacterial Culture Supernatant for Elucidation of Immune Modulators in the Environment

Nucleotide-binding oligomerization domain protein 1 (Nod1) is an intracellular protein involved in recognition of the bacterial component peptidoglycan. This recognition event induces a host defense response to eliminate invading pathogens. The genetic variation of Nod1 has been linked to several inflammatory diseases and allergies, which are strongly affected by environmental factors. We have found that many of the bacteria that contain DAP-type peptidoglycan release Nod1 ligands into the environment. However, the structures of natural Nod1 ligands in the environment are not well understood. Herein, we report the isolation and structural elucidation of natural human Nod1 (hNod1) ligands from the Escherichia coli K-12 culture supernatant. The supernatant was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC), resulting in the isolation of several hNod1 stimulatory fractions. Structural characterization studies demonstrated that the molecular structure of the most active fraction was the native hNod1 ligand GlcNAc-(β1–4)-(anhydro)MurNAc-l-Ala-γ-d-Glu-meso-DAP. We also found other peptidoglycan fragments using the 7-(diethylamino)coumarin-3-carbonyl labeling method to enhance sensitivity in mass spectroscopy studies. These results suggested that DAP-containing bacteria release certain hNod1 ligands to the environment, and these ligands would accumulate in the environment and regulate the immune system through Nod1.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.     

High Immunogenicity of the Human Leukocyte Antigen Peptidomes of Melanoma Tumor Cells

Human leukocyte antigens (HLA) bind peptides generated by limited proteolysis in cells and present them at the cell surfaces for recognition by T cells. Through this antigen presentation function they control the specificity of T cell responses and thereby adaptive immune responses. Knowledge of HLA-bound peptides is thus key to understanding adaptive immunity and to the development of vaccines and other specific immune intervention strategies. To gain insight into the antigenicity of melanomas, peptides were extracted from HLA isolated from the tumor cells, separated by two-dimensional HPLC, and sequenced by mass spectrometry. The spectra were analyzed by database-dependent MASCOT searches and database-independent de novo sequencing and, where required, confirmed with synthetic peptides, which were also used to determine their immunogenicity. Comparing four different melanoma cell lines, little overlap of the HLA-bound peptides was found, suggesting a high degree of individualization of the HLA peptidomes. This notwithstanding, the peptidomes were highly immunogenic in the patients from whom the tumor cells had been established and in unrelated patients. This broad cross-patient immunogenicity was only exceptionally related to individual peptides. The majority of the identified epitopes were derived from low to medium abundance proteins, mostly involved in sensitive cellular processes such as cell cycle control, DNA replication, control of gene expression, tumor suppressor function, and protein metabolism. The peptidomes thus provide insights into processes potentially related to tumorigenesis. Furthermore, analyses of the peptide sequences yield information on the specificity of peptide selection by HLA applicable to the developing prediction algorithms for T cell epitopes.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex_8–16GlcNAc₂ modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex₉GlcNAc₂ at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.