Modulation of Natural HLA-B*27:05 Ligandome by Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 2 (ERAP2)

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Highlights

• •HLA-B*27:05 and ERAP2 are risk factors for ankylosing spondylitis.
• •The effects of ERAP2 on the B*27:05 ligandome are defined.
• •P1, P2, P3, P7, and PΩ peptide positions are influenced by ERAP2.
• •These effects provide a basis for the association of ERAP2 with the autoimmune disease.

     

Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo

The association of ERAP1 with ankylosing spondylitis (AS)¹ among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 interaction is a variant-dependent alteration in the balance between epitope generation and destruction determined by the susceptibility of N-terminal flanking and P1 residues to trimming. ERAP1 polymorphism associated with AS susceptibility ensured efficient peptide trimming and high HLA-B27 stability. Protective polymorphism resulted in diminished ERAP1 activity, less efficient trimming, suboptimal HLA-B27 peptidomes, and decreased molecular stability. This study demonstrates that natural ERAP1 polymorphism affects HLA-B27 antigen presentation and stability in vivo and proposes a mechanism for the interaction between these molecules in AS.The mechanism underlying the strong association of HLA-B27 with ankylosing spondylitis (AS) remains unknown. Three main possibilities, each one based on a different molecular feature of HLA-B27, are currently being investigated. The arthritogenic peptide hypothesis (1), based on the canonic antigen-presenting properties of Major Histocompatibility Complex class I (MHC-I) molecules, assumes that a peptide epitope of external origin would activate HLA-B27-restricted T-cells, whose cross-reactivity with a self-derived HLA-B27 ligand would result in autoimmune damage. The misfolding hypothesis (2) is based on the slow folding and tendency to misfold of HLA-B27 (3, 4). An accumulation of misfolded heavy chains (HCs) in the endoplasmic reticulum (ER) would elicit an unfolded protein response and activate pro-inflammatory pathways. The surface homodimer hypothesis (5, 6) is based on the expression of HLA-B27 HC homodimers at the cell surface and their recognition by leukocyte receptors (7), which leads to immunomodulation of inflammatory responses. Because the constitutive binding of endogenous peptides by MHC-I molecules determines not only their antigen-presenting specificity, but also their folding and stability, it was proposed that the HLA-B27 peptidome, through its global influence on the biological behavior of the molecule, is critical to its pathogenetic role (8). This idea found strong support with the discovery of the association of ER aminopeptidase (ERAP) 1 with AS (9) in HLA-B27-positive, but not B27-negative, disease (10). With an estimated population attributable risk of 26%, ERAP1 is the non-MHC gene most strongly associated with AS. Given that ERAP1 is involved in the N-terminal trimming of peptides to their optimal size for MHC-I binding (11–13), its association with AS suggests a pathogenetic mechanism of functional interaction with HLA-B27 that influences peptide binding and antigen presentation. ERAP1 trimming is limited by peptide size, becoming highly inefficient for 8-mers and shorter peptides (13, 14). This is a seemingly unique feature of ERAP1 that is not even shared by its analog ERAP2 (14, 15). The only putative exception, which has not been entirely ruled out, might be insulin-regulated amino peptidase (IRAP), an endosomal analog of ERAP1 involved in cross-presentation, but probably not in processing of constitutive MHC-I ligands (16, 17). IRAP degrades peptides to smaller products than ERAP1 in vitro (18). The three-dimensional structure of ERAP1 reveals a substrate binding cavity close to the catalytic site, as well as four domains; the conformational rearrangement between an open and a closed conformation, presumably induced upon substrate binding, regulates its enzymatic activity (19, 20). The polymorphic residues found among natural ERAP1 variants (21), and often co-occurring in complex allotypes, are located in various topological regions, including some in close proximity to the catalytic site, the substrate binding cavity, or domain junctions. Therefore, they might alter ERAP1 activity by directly affecting catalysis, altering substrate binding, or modulating domain rearrangements. The association of ERAP1 with AS does not by itself reveal the specific feature(s) determining the pathogenetic role of HLA-B27. Indeed, ERAP1 might influence the generation of specific pathogenetic epitopes; have a general effect on the HLA-B27 peptidome, altering the stability or other features of the molecule; or both. This study investigated general effects of ERAP1 polymorphism on the HLA-B27 peptidome by comparing the size distribution, molecular features, and N-terminal flanking sequences of peptides from human cells expressing the AS-associated B*27:04 subtype and different natural variants of ERAP1.     

Identification of an Unconventional Subpeptidome Bound to the Behçet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)

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Highlights

• •A novel HLA-ABC-triple knockout cell model to study the HLA-B*51 peptidome.
• •Enrichment of the unconventional non-Pro/Ala2 HLA-B*51 peptides following ERAP1 silencing.
• •Knockdown of ERAP1 increases the length of non-Pro/Ala2 and Ala2 peptides, not that of Pro2.
• •ERAP1 regulation of HLA-B*51 cell surface expression is cell type dependent.

     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.     

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphism Relevant to Inflammatory Disease Shapes the Peptidome of the Birdshot Chorioretinopathy-Associated HLA-A*29:02 Antigen

Birdshot chorioretinopathy is a rare ocular inflammation whose genetic association with HLA-A*29:02 is the highest between a disease and a major histocompatibility complex (MHC) molecule. It belongs to a group of MHC-I-associated inflammatory disorders, also including ankylosing spondylitis, psoriasis, and Behçet''s disease, for which endoplasmic reticulum aminopeptidases (ERAP) 1 and/or 2 have been identified as genetic risk factors. Since both enzymes are involved in the processing of MHC-I ligands, it seems reasonable that common peptide-mediated mechanisms may underlie the pathogenesis of these diseases. In this study, comparative immunopeptidomics was used to characterize >5000 A*29:02 ligands and quantify the effects of ERAP1 polymorphism and expression on the A*29:02 peptidome in human cells. The peptides predominant in an active ERAP1 context showed a higher frequency of nonamers and bulkier amino acid side chains at multiple positions, compared with the peptides predominant in a less active ERAP1 background. Thus, ERAP1 polymorphism has a large influence, shaping the A*29:02 peptidome through length-dependent and length-independent effects. These changes resulted in increased affinity and hydrophobicity of A*29:02 ligands in an active ERAP1 context. The results reveal the nature of the functional interaction between A*29:02 and ERAP1 and suggest that this enzyme may affect the susceptibility to birdshot chorioretinopathy by altering the A*29:02 peptidome. The complexity of these alterations is such that not only peptide presentation but also other potentially pathogenic features could be affected.Several major histocompatibility complex class I (MHC-I)¹ alleles are strongly associated with polygenic inflammatory diseases, including birdshot chorioretinopathy (BSCR: A*29:02), ankylosing spondylitis (AS: HLA-B*27), psoriasis (C*06:02), and Behçet''s disease (HLA-B*51). In the three latter disorders, ERAP1, an aminopeptidase of the endoplasmic reticulum performing the final trimming of MHC-I ligands (1, 2), is also a risk factor and is in epistasis with the predisposing MHC-I allele (3–5). These studies suggest common pathogenetic mechanisms involving the MHC-I bound peptidome. ERAP2, a related enzyme that acts in concert with ERAP1 (6, 7), influences the susceptibility to BSCR (8), AS (although not necessarily in epistasis with HLA-B*27) (9), Crohn′s disease (10), and preeclampsia (11–13).BSCR is a rare and severe form of bilateral posterior uveitis, showing a progressive inflammation of the choroid and retina, whose association with HLA-A*29 is the strongest for any disease and MHC. The frequency of this allele is about 7% in healthy individuals but >95% in BSCR patients (14, 15). This association specifically concerns A*29:02 and not the closely related allotype A*29:01 (8).Genetic studies on BSCR also showed a highly significant association within the LNPEP gene (rs7705093) in the 5q15 region, which includes the ERAP1 and ERAP2 genes. One single nucleotide polymorphism (SNP) in this region (rs10044354) correlated with ERAP2 expression. This was confirmed at the protein level, leading to the conclusion that ERAP2 expression predisposes to BSCR. Yet, an involvement of functional ERAP1 polymorphisms, not determining protein expression, was not excluded. These polymorphisms have a large influence on the HLA-B*27 peptidome (16, 17). In contrast, the effects of ERAP2 on MHC-I peptidomes are poorly understood and are probably dependent on the particular ERAP1 context since ERAP2 cooperates with ERAP1 in peptide processing. Thus, the present study was conducted to characterize A*29:02-bound peptidomes in various ERAP1 backgrounds and to determine the influence of ERAP1 polymorphism on the amounts and features of A*29:02 ligands in human cells.     

Systematic Identification and Comparative Analysis of Human Cartilage-Derived Self-peptides Presented Differently by Ankylosing Spondylitis (AS)-Associated HLA-B*27:05 and Non-AS-associated HLA-B*27:09

International Journal of Peptide Research and Therapeutics - The human leukocyte antigen B27 (HLA-B27) gene has been observed to significantly increase the risk of developing ankylosing spondylitis...     

Genetic Polymorphisms of Stromal Interaction Molecule 1 Associated with the Erythrocyte Sedimentation Rate and C-Reactive Protein in HLA-B27 Positive Ankylosing Spondylitis Patients

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The development of ankylosing spondylitis is still unclear. Genetics factors such as human leukocyte antigen HLA-B27 and ERAP1 have been widely reported to associate to AS susceptibility. In this study, we enrolled 361 AS patients and selected four tagging single nucleotides polymorphisms (tSNPs) at STIM1 gene. The correlation between STIM1 genetic polymorphisms and AS activity index (BASDAI, BASFI, BAS-G) as well as laboratory parameters of inflammation (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)) were tested. Our results indicated that HLA-B27 positive AS patients who are carrying the minor allele homozygous G/G genotype of SNP rs3750996 significantly associated with a higher level of ESR in serum. Furthermore, rs3750996/rs3750994 pairwise allele analysis indicated that G-C haplotypes also significantly correlated with higher level of ESR as well as CRP. These findings provide a better understanding of STIM1 genetic contribution to the pathogenesis of AS.     

UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.     

Association Study between the FTCDNL1 (FONG) and Susceptibility to Osteoporosis

Osteoporosis is a systemic skeletal disease characterized by a decreased bone mineral density that results in an increased risk of fragility fractures. Previous studies indicated that genetic factors are involved in the pathogenesis of osteoporosis. Polymorphisms of the FONG (FTCDNL1) gene (rs7605378) were reported to be associated with the risk of osteoporosis in a Japanese population. To assess whether polymorphisms of the FTCDNL1 gene contribute to the susceptibility and severity of osteoporosis in a Taiwanese population, 326 osteoporosis patients and 595 controls of a Taiwanese population were included in this study. Our results indicated that rs10203122 was significantly associated with osteoporosis susceptibility among female. Our findings provide evidence that rs10203122 in FTCDNL1 is associated with a susceptibility to osteoporosis.     

Retention in Endoplasmic Reticulum 1 (RER1) Modulates Amyloid-β (Aβ) Production by Altering Trafficking of γ-Secretase and Amyloid Precursor Protein (APP)

The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615–29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.     

Genetic Susceptibility to Refractive Error: Association of Vasoactive Intestinal Peptide Receptor 2 (VIPR2) with High Myopia in Chinese

Myopia is the most common ocular disease worldwide. We investigated the association of high myopia with the common single nucleotide polymorphisms (SNPs) of five candidate genes – early growth response 1 (EGR1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), jun oncogene (JUN), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide receptor 2 (VIPR2). We recruited 1200 unrelated Chinese subjects with 600 cases (spherical equivalent ≤−8.00 diopters) and 600 controls (spherical equivalent within ±1.00 diopter). A discovery sample set was formed from 300 cases and 300 controls, and a replication sample set from the remaining samples. Tag SNPs were genotyped for the discovery sample set, and the most significant haplotypes and their constituent SNPs were followed up with the replication sample set. The allele and haplotype frequencies in cases and controls were compared by logistic regression adjusted for sex and age to give P _a values, and multiple comparisons were corrected by permutation test to give P _aemp values. Odd ratios (OR) were calculated accordingly. In the discovery phase, EGR1, JUN and VIP did not show any significant association while FOS and VIPR2 demonstrated significant haplotype association with high myopia. In the replication phase, the haplotype association for VIPR2 was successfully replicated, but not FOS. In analysis combining both sample sets, the most significant association signals of VIPR2 were the single marker rs2071625 (P _a = 0.0008, P _aemp = 0.0046 and OR = 0.75) and the 4-SNP haplotype window rs2071623-rs2071625-rs2730220-rs885863 (omnibus test, P _a = 9.10e-10 and P _aemp = 0.0001) with one protective haplotype (GGGG: P _aemp = 0.0002 and OR = 0.52) and one high-risk haplotype (GAGA: P _aemp = 0.0027 and OR = 4.68). This 4-SNP haplotype window was the most significant in all sample sets examined. This is the first study to suggest a role of VIPR2 in the genetic susceptibility to high myopia. EGR1, JUN, FOS and VIP are unlikely to be important in predisposing humans to high myopia.     

Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 Interacts with TGD1, TGD2, and TGD4 to Facilitate Lipid Transfer from the Endoplasmic Reticulum to Plastids

The biogenesis of photosynthetic membranes in the plastids of higher plants requires an extensive supply of lipid precursors from the endoplasmic reticulum (ER). Four TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins (TGD1,2,3,4) have thus far been implicated in this lipid transfer process. While TGD1, TGD2, and TGD3 constitute an ATP binding cassette transporter complex residing in the plastid inner envelope, TGD4 is a transmembrane lipid transfer protein present in the outer envelope. These observations raise questions regarding how lipids transit across the aqueous intermembrane space. Here, we describe the isolation and characterization of a novel Arabidopsis thaliana gene, TGD5. Disruption of TGD5 results in similar phenotypic effects as previously described in tgd1,2,3,4 mutants, including deficiency of ER-derived thylakoid lipids, accumulation of oligogalactolipids, and triacylglycerol. Genetic analysis indicates that TGD4 is epistatic to TGD5 in ER-to-plastid lipid trafficking, whereas double mutants of a null tgd5 allele with tgd1-1 or tgd2-1 show a synergistic embryo-lethal phenotype. TGD5 encodes a small glycine-rich protein that is localized in the envelope membranes of chloroplasts. Coimmunoprecipitation assays show that TGD5 physically interacts with TGD1, TGD2, TGD3, and TGD4. Collectively, these results suggest that TGD5 facilitates lipid transfer from the outer to the inner plastid envelope by bridging TGD4 with the TGD1,2,3 transporter complex.     

Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)

LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca²⁺-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca²⁺ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca²⁺ concentrations. Our results suggest that compartmental changes in Ca²⁺ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.