key: cord-0329393-grpndbtt
authors: Mattorre, Benedetta; Caristi, Silvana; Donato, Simona; Volpe, Emilia; Faiella, Marika; Paiardini, Alessandro; Sorrentino, Rosa; Paladini, Fabiana
title: A short ERAP2 that binds IRAP is expressed in macrophages independently from gene variation
date: 2022-03-07
journal: bioRxiv
DOI: 10.1101/2022.03.07.483178
sha: 947b1b0a12fd78bb0b97395e5e6de9ee34acbfd0
doc_id: 329393
cord_uid: grpndbtt

The M1 zinc metalloproteases ERAP1, ERAP2 and IRAP play a role in HLA-I antigen presentation by refining the peptidome either in the ER (ERAP1 and ERAP2) or in the endosomes (IRAP). They have been also entrusted with other, although less defined, functions such as the regulation of the angiotensin system and blood pressure. In humans, ERAP1 and IRAP are commonly expressed. ERAP2 instead has evolved under balancing selection that maintains two haplotypes one of which undergoing RNA splicing leading to nonsense-mediated decay and loss of protein. Hence, likewise in rodents in which the ERAP2 gene is missing, about a quarter of the human population does not express ERAP2. We report here that macrophages, but not monocytes or other mononuclear blood cells, express and secrete an ERAP2 shorter form independently from the haplotype. The generation of this “short” ERAP2 is due to an autocatalytic cleavage within a distinctive structural motif and requires an acidic microenvironment. Remarkably, ERAP2 “short” binds IRAP and the two molecules are co-expressed in the endosomes as well as in the cell membrane. Of note, the same phenomenon could be observed in some cancer cells. These data prompt to reconsider the role of ERAP2 which might have been maintained in humans because fulfilling a relevant function as “short” form in specialized cells.

The aminopeptidases ERAP1, ERAP2 and IRAP, the latter being the product of the gene 55 LNPEP, are members of the oxytocinase subfamily of M1 Zinc-metallopeptidases whose 56 corresponding genes lie contiguously on chromosome 5 (1,2). ERAP1 and ERAP2 reside 57 in the ER where they co-operate to trim the N-terminal peptide residues to the correct 58 length to bind the HLA-I molecules (3). IRAP instead, thanks to an additional N-terminal 59 cytoplasmic domain, is retained in the endosomal vesicles from where it can traffic to the 60 cell membrane forming a type II integral membrane glycoprotein (4). IRAP is a 61 multifaceted protein: it has been shown to be involved in cross-presentation in the 62 endosomes of dendritic cells (DC) and, when in the cell membrane, to catalyze the final 63 step of the angiotensinogen to angiotensin IV (AT4) conversion being itself a receptor for 64 AT4 (5,6). In addition, it has been shown to be involved in several other functions, ranging 65 from the insulin metabolic pathway to vesicular trafficking and even in cognitive processes 66 (7,8). Furthermore, the three genes have been found associated with autoimmune and 67 inflammatory conditions, hypertension, and cancer (2, 9-15). Since they are regulators of 68 the Renin-Angiotensin system (RAS), their imbalance can have indeed consequences on 69 different aspects of the associated pathologies (16) (17) (18) . In the MHC-I-opathies (19) , while 70 ERAP1 association resides in variants influencing its trimming activity, the molecular terms 71 of the association of ERAP2 and LNPEP with some of these diseases are at present 72 poorly understood (20). In particular, ERAP2 has been shown to be involved in MHC-I- 73 opathies and other conditions, while much of its biology remains uncertain, including the 74 dramatic variability in its functional opposite haplotypes maintained in the population. This 75 is mainly due to a balanced polymorphism at SNP rs2248374 that makes one haplotype 76 4 null due to a shift in the splicing that leads to mRNA instability and non-sense mediated 77 decay (NMD). Consequently, about a quarter of the human population does not express 78 ERAP2 full length and about 50% shows an allelic exclusion. Of note, this gene is missing 79 in rodents as well as in several other species (2, 21). These observations raise several 80 questions about the physiological role of ERAP2. Indeed, by looking at the expression of 81 ERAP1, ERAP2 and IRAP along the zoological scale, we have recently pointed out how a 82 high degree of redundancy and, most likely interchangeability, characterizes these three 83 aminopeptidases (2). ERAP1 has been shown to be secreted (22) and IRAP has also a 84 soluble counterpart detectable in the serum of pregnant women (23). More recently, an 85 ERAP2 soluble isoform has been described as released by human monocyte-derived 86 macrophages (MDMs) in response to IFNγ/LPS stimulation and this corresponds to an 87 increased CD8+ T cells activity (24) . With the aim to gain additional information about the 88 interconnections among the three aminopeptidases, we have investigated their expression 89 and interactions in different contexts. Remarkably, we observed an ERAP2 shorter subunit 90 expressed by macrophages as well as by some cancer cells and which forms a complex 91 with IRAP. This ERAP2 "short" corresponds to a N-terminal fragment that requires an 92 acidic microenvironment to be generated and it is present even in those subjects expected 93 to be ERAP2-deficient. These suggestive findings prompted us to reconsider the role of 94 ERAP2 in physiology as well as in pathology. as G/G at rs2248374, indicates that it is the "short" and not the full length ERAP2 that is 272 present in the cell membrane. In addition, the co-expression of the MS4A4A and ERAP2 273 strongly suggests that the "short" ERAP2 is secreted by the M2-type macrophages since, 274 in our hands, MS4A4A-positive cells are present also in the putative M1-type. 275 The confocal analysis of the three aminopeptidases on MDMs ( Figure 1D 

These data indicate that, independently from the ERAP2 genotype, MDMs express a 285 "short" ERAP2 that co-localizes with IRAP and that it is secreted primarily by the M2-type 286 macrophages. respectively. The expression of ERAP1, ERAP2 and IRAP was therefore analyzed by 295 immunofluorescence ( Figure S3 ) and by western blotting (Figure 2A ). Compared to the 296 untreated cells, PMA-treated cells show a higher expression of MS4A4A and, in parallel, of 297 ERAP2 whereas ERAP1 and IRAP did not vary ( Figure S3 ). Western blot analysis 298 (Figures 2A and 2B) showed that U937 cells, despite being genotyped as G/G at SNP shown). Once again, these data confirm that the "short" ERAP2 colocalizes with IRAP. Figure 4A second lower panel, lane 3) . No co-immunoprecipitation 347 between ERAP1 and either ERAP2 or IRAP was detectable ( Figure 4A ). As control, in 348 each box the three MoAbs used were run in the last three lanes to exclude that the heavy 349 chain of the MoAbs used in the immunoprecipitation assay, whose MW is ~50kDa, could Figure 4B ). We concluded that the ~55kDa is generated from the 360 full-length protein through a mechanism that requires acidic conditions. 361 ERAP2 contains a potential autocatalytic cleavage site 362 In order to gain structural insights into the mechanism of the cleavage observed in ERAP2 363 but not in the closely related ERAP1 and IRAP, the crystal structure of ERAP2 (PDB: 364 5AB0) was investigated and compared to the other two aminopeptidases (39). In the case 365 of ERAP2, the protein structure shows the presence of a unique extended loop, 366 encompassing residues 500-520 ( Figure 4C ). This distinctive structural motif is peculiar of 367 ERAP2 and contains two potential sites, which could be sensitive to low pH regulation: 1) 368 a disulfide bridge formed by Cys 503 and Cys 514, which might be broken at low pH 369 values and 2) a specific Asp-Pro motif ( Figure 4D ). However, while the disulfide bridge is 370 also conserved in ERAP1, the Asp-Pro motif is unique of ERAP2. This site has been 371 reported to be particularly sensitive to pH variations (40) and it could represent a weak 

An open question is from which precursor the ~55kDa fragment is generated. Indeed, 386 while in individuals typed as "A" at rs2248374 as well in the case of the U937 cells, it is 387 likely to derive from the full-length protein (Figures 1 and 4) , in the case of the "G" 388 haplotype we could not detect any precursor in macrophages. A possible explanation is 389 that in macrophages, part of the ERAP2 mRNA copies can escape NMD and the naturally 390 encoded truncated protein which normally undergoes proteolysis, can be stabilized, 391 possibly by the binding to IRAP, when an acidic microenvironment allows the generation of 392 the ~55kDa fragment. To explore this hypothesis, two constructs were generated (Figure 393 S1): one carrying the natural "G" mRNA which encodes for the specific sequence that 394 stops at amino acid 532 and carries the sequence VRIKRVTE at C-terminus before the 395 stop codon and the other carrying the alternative "A" mRNA but interrupted at the same 396 length as the "G" haplotype and therefore differing at the C-terminus for the last amino 397 acids (LAFLGENAEVK) (Figure 6 ). Following transfection into HEK293T cells, both 398 proteins were expressed. However, the results show that, at low pH, the "short" ERAP2 is 399 obtained only from the construct carrying the "VRIKRVTE" motif encoded by the "G" 400 haplotype (Fig.6A) . Modelling of the two truncated proteins support our findings: in the Figure 6B ). 408 We concluded that the specific C-terminus of the "G" isoform was responsible, in specific 418 We report here the existence of a short form of ERAP2 that binds IRAP and is secreted by 419 the M2-type macrophages. Remarkably, this "short" ERAP2 is generated by an 420 autocatalytic process that occurs in an acidic microenvironment. A similar event has been 421 reported for MUC2 protein with which ERAP2 shares some features. Indeed, ERAP2 422 displays a unique disordered loop containing two cysteines (Cys503 and Cys514) and a 423 close downstream Asp517-Pro518 specific sequence. The reduction of the disulfide bridge 424 could expose and allow the hydrolysis of the Asp-Pro bond as previously described for the 425 MUC2 in the late secretory pathway (40). It is known that IRAP lies in the endosomes 426 whose acidic microenvironment can eventually determine the generation of the ERAP2 427 "short" fragment. We do not know, however, where exactly the encounter between ERAP2 428 and IRAP occurs: i.e. whether ERAP2 reaches the endosomes along its way to the cell 429 membrane or whether it is endocytosed once bound to IRAP. Whatever the case, it is 430 shown here that the two molecules are co-expressed in the endosomes suggesting that it 431 is their acidic microenvironment that allows the catalytic process leading to the "short" 432 ERAP2. In addition, the immunoprecipitation experiments, supported by the modelling, 433 unambiguously demonstrate the existence of a direct, strong binding between the two 434 molecules. It is interesting that the same phenomenon was observed in some cancer cell 435 lines expressing the "short" ERAP2 although the secretion appears to be specific for the 436 MDMs. Most remarkable however is the observation that, differently from the full-length 437 protein, the "short" ERAP2 is expressed in macrophages independently from the 438 polymorphism at rs2248374 that discriminates the two possible haplotypes. While this is 439 conceivable in the haplotype expressing the full length ERAP2 (allele A at rs2248374), we as from two monocytes/macrophages series (unpublished). No RNA corresponding to the 451 ~55kDa ERAP2 described here was found while the ERAP2 Iso/3, although listed, was 452 undetectable in the U937 cells and barely detectable in monocytes and in the M1 but not in 453 the M2 macrophages. These observations, together with the fact that the "short" ERAP2 is 454 obtained by the acid-treatment of the cell lysates, strongly supports its catalytic origin.

Of note, our results also show that the process is distinctive of the differentiated myeloid 456 cells within the PBMCs pointing to a specific role in these cells. It is known that IRAP binds 457 with high affinity AT4, a biologically active fragment of Angiotensin II. IRAP is also named 458 oxytocinase since it was found to regulate the level of circulating oxytocin during the later 459 stages of human pregnancy and, interestingly, is also highly expressed in brain regions 460 associated with cognition being recognized as a potential target for the treatment of 461 cognitive disorders (44). The observation reported here that it can bind ERAP2 adds a 462 novel piece to the intriguing puzzle regulating the IRAP multifaceted functions, including 463 inflammation. In particular, it has been shown that ACE2 (Angiotensin I-converting 464 enzyme-2) is the receptor for SARS-COV-2 (45) and it has been postulated that its 465 reduction in the cell membrane due to the endocytic pathway associated with SARS-CoV-466 2 binding, can play a role in altering the inflammatory balance (16) . ERAP2 "short" 467 20 fragment secreted by type 2 macrophages, by binding IRAP is likely to interfere with the 468 RAS (Renin-Angiotensin-System) by modulating its anti-inflammatory arm. In this context, 469 a polymorphism in the ERAP2 gene has been found associated with death due to SARS- 

ERAP2 carrying either the LAFLGENA or the 632 VRIKRVTE motif. The initial atomic coordinates were taken from the X-ray crystal structure 633 of ERAP2 (Protein Data Bank ID: 5AB0). The Python protocol described in Yang et al. 634 (32), which makes use of the Rosetta 3.13 neural network to generate inter-residue 635 distance and orientation constraints for a sequence of unknown structure

Rosetta minimizer was then used to find the backbone conformation consistent with the

The oxytocinase subfamily of M1 aminopeptidases

The Multifaceted Nature 653 of Aminopeptidases ERAP1, ERAP2, and LNPEP: From Evolution to Disease

Peptidases trimming MHC 656 class I ligands

IRAP 658 identifes an endosomal compartment required for MHC class I cross-presentation

Is There an Interplay Between the 664 Functional Domains of IRAP?

The 667 Role of Insulin Regulated Aminopeptidase in Endocytic Trafficking and Receptor Signaling 668 in Immune Cells

Identification and 671 characterization of a new cognitive enhancer based on inhibition of insulin-regulated 672 aminopeptidase

Role as Predictive Renal Injury Biomarkers

Understanding the Pathogenesis of Spondyloarthritis

The 679 roles of ERAP1 and ERAP2 in autoimmunity and cancer immunity: New insights and 680 perspective

The 682 Impact of the 'Mis-Peptidome' on HLA Class I-Mediated Diseases: Contribution of ERAP1 683 and ERAP2 and Effects on the Immune Response

Genetic Study on Small Insertions 686 and Deletions in Psoriasis Reveals a Role in Complex Human Diseases

Vasopressin inactivation: Role of insulin-689 regulated aminopeptidase

Regulation of ERAP1 and ERAP2 genes and their 692 disfunction in human cancer

ERAP1 and 695 ERAP2 Enzymes: A Protective Shield for RAS against COVID-19?

Endoplasmic Reticulum Aminopeptidase 2, a common immunological link to 698 adverse pregnancy outcomes and cancer clearance?

Association of ERAP2 gene variants with risk of pre-eclampsia 701

among Iranian women

MHC-I-opathy-unified 703 concept for spondyloarthritis and Behçet disease

Endoplasmic reticulum aminopeptidases in the 706 pathogenesis of ankylosing spondylitis

Balancing 709 selection maintains a form of ERAP2 that undergoes nonsense-mediated decay and 710 affects antigen presentation

Secretion of endoplasmic reticulum 713 aminopeptidase 1 is involved in the activation of macrophages induced by 714 lipopolysaccharide and interferon-gamma

Ectodomain shedding of cystinyl aminopeptidase from human 717 placental membranes

Reticulum Associated Aminopeptidase 2 (ERAP2) Is Released in the Secretom

Activated MDMs and Reduces in vitro HIV-1 Infection

An allelic variant 723 in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression 724 of the two genes

Comparison of protein precipitation methods for various rat brain structures prior to 727 proteomic analysis

Image to ImageJ: 25 years of image 729 analysis

Quantitative Colocalization Analysis of Multicolor 731

Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological 732 Phenomena

Crystal structures 734 of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-735 terminal peptide trimming

Structural Basis 738 for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum

Aminopeptidase 2

Crystal 741 structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides

The HADDOCK web server for data-driven 744 biomolecular docking

Contact-based prediction of binding affinity in protein-746 protein complexes

PRODIGY: a web-server 748 for predicting the binding affinity in protein-protein complexes

Improved protein 751 structure prediction using predicted interresidue orientations

The 754 macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell-mediated 755 resistance to metastasis

Endoplasmic Reticulum Aminopeptidases in EBV-B Cell Lines from Healthy Donors

MS4A4A: a novel cell 762 surface marker for M2 macrophages and plasma cells

The Role of Conformational Dynamics in Antigen 765 Trimming by Intracellular Aminopeptidases

An autocatalytic cleavage in the C 768 terminus of the human MUC2 mucin occurs at the low pH of the late secretory pathway

ERAP2/Iso3 Isoform Expression Is Triggered by Different Microbial Stimuli in Human 772

Could It Play a Role in the Modulation of SARS-CoV-2 Infection?

Genetic 775 analysis of isoform usage in the human anti-viral response reveals influenza-specific 776 regulation of ERAP2 transcripts under balancing selection

RNA editing 779 signature during myeloid leukemia cell differentiation

Aminopeptidase (IRAP) Inhibitors: A Literature Review

Molecular 785 interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

Genetic risk factors for death with SARS-788

The interplay 790 between immunosenescence and age-related diseases