The Antigen-Processing Pathway via Major Histocompatibility Complex I as a New Perspective in the Diagnosis and Treatment of Endometriosis

These enzymes critically shape the MHC-I immunopeptidome. The ERAPs remove the N-terminal residues from the antigenic precursor peptides and generate optimal-length peptides (i.e., 8–10-mers) to fit into the MHC class I groove. Both are Zn-metallopeptidases sharing about 50% amino acid identity (Tsujimoto and Hattori 2005). ERAP1 adopts two conformations: one closed and active and another open and with low activity (Stamogiannos et al. 2015). The transition between both the states is mediated by substrate binding to a regulatory site distinct from the catalytic site, in such a way that short peptides cannot reach the latter. This explains the unique molecular ruler mechanism of ERAP1—peptides of 9–16 residues, but not shorter, are efficiently trimmed (Chang et al. 2005). Both ERAPs, however, cannot trim peptide bonds involving proline. ERAPs can destroy the putative HLA class I ligands by reducing the length of antigenic peptides below the threshold of 8–10 amino acids when they are overactive. A study by Saveanu et al. (2005) indicated that ERAP1 and ERAP2 were colocalized in vivo and linked in complexes. This implies a concerted peptide trimming by the ERAP1 and ERAP2 complexes in the ER. On the other hand, the loss of both enzymes is a frequent event significantly associated with the lack of HLA class I surface expression (Hammer et al. 2007).

ERAP1/ERAP2 protein expression is detected in many tissues and is induced by type I and type II interferons (IFNs) and TNF-α, suggesting that they are essential for immune control (Saric et al. 2002; Lee 2017).

The human ERAP1 and ERAP2 genes are located on chromosome 5q15 in opposite orientations (Lee 2017). Both genes are polymorphic with strong linkage disequilibrium (LD) across the chromosome 5q15 locus, and many functional variants appear to affect their enzymatic activity and the expression level or both. ERAP1 variants are complex allotypes including multiple nonsynonymous single nucleotide polymorphisms (SNPs), known as haplotypes. Ten ERAP1 haplotypes (Hap1 to Hap10) account for over 99% of the ERAP1 variants in human populations (Reeves et al. 2013; Ombrello et al. 2015). Polymorphic amino acids are located near the catalytic site (residues 346, 349), in the peptide-binding site (residues 725 and 730) or in the interdomain regions or other locations that can affect the conformational changes associated with enzymatic activity (residues 528 and 575). Therefore, these polymorphisms may influence ERAP1 activity in multiple ways (Hanson et al. 2018; López de Castro 2018).

In contrast to ERAP1, nonsynonymous changes affecting the amino acid sequence of ERAP2 seem to be limited. The polymorphism rs2549782 coding for the K392N (Lys392Asp) change affects the ERAP2 activity. The N392 allele is in LD with the G allele of rs2248374 (allotype B), a polymorphism that favors nonsense-mediated RNA decay and impairs protein expression (Andrés et al. 2010). As a result, only the K392 variant (allotype A—rs2248374A) is expressed in most populations (Evnouchidou et al. 2012). Because both alleles of rs2248374 occur with a similar frequency in the population due to balancing selection, about 25% of individuals fail to express ERAP2. However, ERAP2 short protein isoforms of unknown function were detected in virus-infected rs2248374 GG individuals (Ye et al. 2018; Saulle et al. 2020). It should be noted that homozygotes from the B allotype have reduced levels of MHC class I expression on B cell surfaces (Andrés et al. 2010). This indicates that the rs2248374 polymorphism in the ERAP2 gene influences the expression of MHC class I on the cells of the immune system. Moreover, Lospinoso et al. (2021) found that the ERAP2-asparagine (N) isoform, expressed in the trophoblast cell line JEG-3, significantly activates the immune cells for killing by both cytotoxic T cells and NK cells, which may determine the success of pregnancy.

Other polymorphisms, such as rs10044354, or SNPs in strong LD with it, may influence the expression of ERAP2 (Kuiper et al. 2014). A polymorphism located in the ERAP2 promoter region, rs7586269, was associated with opposite changes in the expression of ERAP1 and ERAP2 so that decreased expression of ERAP2 correlated with increased ERAP1 expression, suggesting a concerted regulation of the expression of both genes by this SNP (Paladini et al. 2018).

ERAP1 and ERAP2 polymorphisms and expression induce significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and reflect the separate roles in their processing of the MHC-I ligands (López de Castro 2018). Perhaps the polymorphism of these genes influencing the expression of proteins, their activity, and substrate specificity may be related to a predisposition to endometriosis. The inability to form the correct HLA class I complexes with the appropriate peptides may result in a lack of immune response by CD8⁺ lymphocytes and NK cells, contributing to the attachment of the ectopic endometrium to various sites of female organs. To date, there are no studies on ERAP polymorphism and expression in ectopic endometrium tissues such as the peritoneum, ovaries, and fallopian tubes. Therefore, it remains unclear whether this variation influences disease development, its association with the location of endometriotic lesions, and, subsequently, its connection to disease severity. The only pregnancy-related disease in which the role of ERAP has been studied is preeclampsia (PE). Xu et al. (2020) revealed that ERAP1 expression was significantly elevated in placental tissues of PE, and hypoxia increased ERAP1 expression in trophoblast. Moreover, ERAP2 is also a good candidate to contribute to the development of PE and other pregnancy-related diseases because of its involvement in the regulation of the immune response, pro-inflammatory cytokine production, and blood pressure. The fetal ERAP2 SNP rs2549782 genotype was associated with a higher risk for PE in African American women (Vanhille et al. 2013). A study by Seamon et al. (2020) revealed that the ERAP1 protein was upregulated in the first-trimester placentae in comparison to placentae at delivery from both normotensive and PE women. In turn, ERAP2 protein expression was similar in normotensive women at delivery compared to expression in the first trimester. Our research also indicated the role of ERAP1 and ERAP2 gene polymorphisms in recurrent implantation failure following in vitro fertilization (Piekarska et al. 2022) and in recurrent miscarriage following natural conception (Wilczyńska et al. 2019). Moreover, an increased level of plasma ERAP2 protein was associated with miscarriage after in vitro fertilization (Piekarska et al. 2021). It should be noted that a large group of these patients were women suffering from endometriosis.

It is quite intriguing that the aminopeptidases ERAP1 and ERAP2 that reside in the ER are released into the peripheral blood. Moreover, studies are demonstrating their ability to trim receptors for pro-inflammatory cytokines such as TNF-α and the type I IL-6 cytokine receptor (IL-6Rα), and the type II IL-1 decoy receptor (IL-1RII) (Cui et al. 2003a,b). Cui et al. (2002) found that ERAP1 can bind to the extracellular domain of the tumor necrosis factor receptor-1 (TNFR1), facilitating its shedding via the formation of a TNFR1/ERAP1 complex. The authors observed that overexpression of ERAP1 causes a production of soluble TNFR1, which competes with TNF receptors on the cell surface, hence attenuating TNF-α bio-activity when ERAP1 levels are increased and restoring TNF-α when levels decrease. TNF receptor shedding may also decrease the number of cell-surface receptors accessible for ligand binding. Therefore, it can be assumed that overexpression of ERAP1 may attenuate the inflammation. Furthermore, reports are showing a multifunctional role for ERAP1 and ERAP2. ERAPs participate in many biological processes including post-natal angiogenesis and regulation of blood pressure (Cifaldi et al. 2012). ERAP1 and ERAP2 cleave Angiotensin II (Ang II) into Ang III and IV (Mattorre et al. 2022).

LNPEP (known also as Insulin-Regulated membrane Aminopeptidase [IRAP], and Placental Leucine Aminopeptidase [PLAP], and Angiotensinogen receptor 4 [ATR4]), with broad tissue distribution, is another aminopeptidase whose role in the pathomechanism of endometriosis has not yet been elucidated. The aminopeptidase is found both in the cytosol and endosomes, where it cleaves proteins before cysteine, leucine, and various other amino acids. Due to an additional N-terminal cytoplasmic domain, LNPEP is retained in the endosomal vesicles from where it can migrate to the cell membrane forming a type II integral membrane glycoprotein. LNPEP co-segregates with the insulin-responsive glucose transporter type 4 (GLUT4) transporter in storage vesicles that traffic to and from the plasma membrane in insulin-responsive cells (Summers et al. 1999; Descamps et al. 2020). LNPEP, located in the membrane, catalyzes the final step of converting angiotensinogen to angiotensin IV. It is also known as the AT4 receptor and is expressed in the human brain, as well as in the heart, kidneys, adrenal glands, and blood vessels. LNPEP has a positive influence on blood flow, neuroprotection, synaptogenesis, long-term potentiation, and memory consolidation and retrieval. LNPEP is also an essential component in the rennin–angiotensin system (Mattorre et al. 2022). LNPEP can be secreted in soluble form in maternal serum during normal pregnancy and plays a role in maintaining homeostasis during pregnancy. LNPEP expression has also been demonstrated by placental cells in pregnancy. It increases progressively with gestational age and, as oxytocinase, is involved in the induction or inhibition of pain during labor (Nonn et al. 2021). LNPEP degrades other peptide hormones, such as vasopressin and angiotensin III (Paladini et al. 2020). Additionally, it is engaged in the generation of antigenic peptides for cross-presentation in endosomal compartments in MHC class I antigen processing (Saveanu et al. 2009; Segura et al. 2009; Weimershaus et al. 2012; Agrawal and Brown 2014).

The LNPEP gene consists of 18 exons (17 introns) on chromosome 5q15 and has two major transcript variants. The LNPEP rs4869317 T/A SNP (located in the intron 1 of the transcript variant 1, RefSeq NM_005575.3) was found to be associated with vasopressinase activity (Nakada et al. 2011). The A609T SNP interacts with the hinge domain III of IRAP. This SNP reduces the enzyme’s activity by almost half (Mpakali et al. 2015).

Tapasin is a V-C1 (variable-constant) immunoglobulin superfamily (IgSF) molecule that links MHC class I molecules to the TAP in the ER. Tsn stabilizes the structure of peptide–MHC-I and promotes the dissociation of low-affinity peptides. Tapasin thus functions as an MHC-I-specific peptide editor or peptide proofreader (Tan et al. 2002; Praveen et al. 2010; Thomas and Tampé 2017, 2019; Margulies et al. 2020). It has up to four MHC class I complexes, and tapasin can bind to a single TAP molecule. This protein contains a C-terminal double lysine motif (KKAE), which is known to maintain membrane proteins in the ER. It has been found that substitutions at position K408 in the transmembrane/cytoplasmic domain of the tapasin affected the expression of MHC class I molecules at the cell surface and downregulated tapasin stabilization of TAP (Petersen et al. 2005).

Tapasin is encoded by the TAPBP gene, located near the MHC at 6p21.3. Alternative splicing results in transcript variants encoding different isoforms (Belicha-Villanueva et al. 2010). Among the SNPs in this gene, it would be worth examining rs3106189 C/T [5′UTR], rs1059288 A/G on 3′UTR, or rs2071888 C/G [T260R]. The first SNP, located in the promoter of the gene among the binding sites for several transcription factors, including interferon regulatory transcription factor (IRF)-1, IRF-2, and IRF-7, could influence its expression level (Shao et al. 2013). The second SNP in 3′UTR might influence microRNA (miRNA) binding. However, rs2071888 C/G (T260R) on exon 4 is in absolute LD with rs1059288 T > C on 3′UTR (Cho et al. 2013).

Over 20 years ago, a gene encoding a Tsn homolog called TAPBPR was discovered that is highly conserved among vertebrates. TAPBPR is an MHC-I dedicated binding protein and has a role in facilitating high-affinity epitope selection and peptide loading in the antigen presentation pathway (Teng et al. 2002; McShan et al. 2022). This protein functions as a second MHC-I-specific chaperone and peptide proofreader. As is already known, a crucial element of MHC-I antigen presentation is the so-called PLC. It is a multisubunit machinery containing an MHC-I-specific chaperone Tsn and TAP. TAPBPR is interferon-γ (IFN-γ)-inducible (like tapasin), recognizes peptide-receptive MHC-I in the ER, and thus catalyzes peptide proofreading, which changes the order of peptides presented on MHC-I on the cell surface. Although TAPBPR has a distinct allomorphic specificity and is independent of PLC, it is thought to share a common catalytic mechanism with Tsn (Thomas and Tampé 2017). The encoded TAPBPR protein—similarly to tapasin—is composed of a signal sequence and three extracellular domains, which include a unique membrane-distal domain, an IgSF V domain and an IgC1 domain, a transmembrane domain, and a cytoplasmatic region (Teng et al. 2002). TAPBPR is now included as a new component of MHC-I antigen presentation but was initially identified with a gene locus that encoded 22% sequence identity with tapasin. TAPBPR interacts with uridine diphosphate (UDP)-glucose: glycoprotein glucosyltransferase 1 (UGGT1), which means it is directly connected to the calreticulin/calnexin cycle. Furthermore, TAPBPR uses a β-hairpin motif that recognizes the peptide-binding status. The β-hairpin, often referred to as the “jack-hairpin,” is situated beneath the floor of the MHC-I peptide-binding groove. Furthermore, it interacts with a conserved residue in β2m, the conformation of which is influenced by the occupancy of MHC-I peptides (Trowitzsch and Tampé 2020).

The human TAPBPR gene is located at chromosome position 12p13.3 next to the paralogous MHC locus, between the CD27 and VAMP1 genes. The TAPBPR gene consists of 7 exons and spans 10 kb. Similar to TAPBP, TAPBPR also possesses TATA and CAATT boxes in its promoter region (Teng et al. 2002). Polymorphisms in MHC class I molecules themselves, as well as in specific components, have a very strong impact on individual disease susceptibility. Thus, it can be speculated that it contributes to the control of pathogens including primarily viral infections. In addition, it may influence the immune system’s recognition of tumors. It is interesting to note that there is a strong association between TAPBPR expression and survival of patients with glioblastoma multiform (Hermann et al. 2015).

TAP is a 150-kDa heterodimeric protein complex (TAP1 and TAP2) and a member of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily of transporters. TAP forms a transmembrane pore in the ER membrane. The opening and closing of these pores depend on ATP binding and hydrolysis. In the ER, together with other elements, it forms a PLC that directs the loading of high-affinity peptides onto nascent MHC-I molecules (Lehnert and Tampé 2017). TAP also plays a pivotal role in transporting peptides into phagosomes and endosomes during cross-presentation in dendritic cells (Mantel et al. 2022). Therefore, TAP constitutes a major component in the adaptive immune response against virally or malignantly transformed cells (Leone et al. 2013; Verweij et al. 2015). TAP was found in all nucleated cells of jawed vertebrates (Lehnert and Tampé 2017).

TAP protein is encoded at chromosomal location 6p21.32 in the MHC class II region between the DQB1 and DPA1 loci (McCluskey et al. 2004). Both TAP1 and TAP2 transcription is upregulated by interferon-β (IFN-β), IFN-γ, and TNF-α (Elliott 1997). A deletion or mutation in the TAP gene severely affects the translocation of peptides to the ER. Therefore, several genetic and protein variants of TAP1 and TAP2 have been associated with autoimmune diseases, cancers, and infections (Lankat-Buttgereit and Tampé 2002; Leone et al. 2013; Qian et al. 2017; Tabassum et al. 2021). So far, no literature data describes the role of TAP proteins and their genetic polymorphism in endometriosis. Twelve alleles of TAP1 and TAP2 have been named by the HLA Nomenclature Committee (http://hla.alleles.org/alleles/classo.html) (Robinson et al. 2015). The functional consequences of the non-synonymous variants rs1057141 (I393V) in TAP1 and rs1800454 (V379I) in TAP2 are unknown. In contrast to this, rs241447 (T665A) present in exon 11 of TAP2 seems to be functionally relevant. One study reported that rs241447 could influence the relative proportion of alternative splicing isoforms of TAP2 differing in efficiency and specificity of transport of different peptides. Interestingly, rs241447 is in complete LD (at least in Caucasians) with the other SNP—rs241456 located in the 3′UTR of the TAP2 gene. According to in vitro studies, this SNP participates in the creation of a potential binding site for hsa-miR-1270 and therefore suppresses TAP2 production in an allelic-specific manner (Wiśniewski et al. 2020).

The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. Proteasomes cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. Among the different types of proteasomes, the immunoproteasome has the function of processing MHC class I peptides (Fricker 2020). There are three catalytic subunits within the immunoproteasome: LMP2, LMP7, and the multicatalytic endopeptidase complex subunit-1 (MECL-1 also called LMP10) responsible for the processing of proteins to peptides. The LMP2 and LMP7 subunits perform chymotrypsin-like activity and cleave at hydrophobic amino acids. LMP10 exhibits trypsin-like activity (Ferrington and Gregerson 2012; Leone et al. 2013). They are induced in the majority of cells by stimulation with IFN-γ, under conditions of oxidative or inflammatory stress (Heink et al. 2005; Shin et al. 2006; Morozov and Karpov 2018). The immunoproteasome produces a different range of peptides than the standard proteasome (Fricker 2020).

The genes encoding LMP2—PSMB9 and LMP7—PSMB8 are located in the MHC class II region on chromosome 6. There are two reported isoforms. These proteins have been shown to have cytoplasmic and nuclear cellular localization. The gene PSMB10 for LMP10 is found outside the MHC class II region on chromosome 16 and is expressed as a single copy. One of the SNPs worth testing is rs17587 A/G [R60H] in exon 3 in the LMP2 protein, which can have a significant impact on immunoproteasome function (Ferrington and Gregerson 2012). It was found that MECL-1 and LMP7 deficiency resulted in reduced MHC class I expression in virus-infected mice (van Helden et al. 2011).

Table 2 summarizes information regarding the molecules involved in antigen processing.