Common allotypes of ER aminopeptidase 1 have substrate-dependent and highly variable enzymatic properties

Polymorphic variation of immune system proteins can drive variability individual responses. Endoplasmic reticulum aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by major histocompatibility complex class I molecules. Coding SNPs in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown affect functional properties the enzyme, but interplay between combinations these as they exist allotypes has not thoroughly explored. We used phased genotype data estimate allotype frequency 2504 individuals across five human populations, generated highly pure recombinant enzymes corresponding ten most common allotypes, systematically characterized their vitro enzymatic properties. find that possess a wide range activities, up 60-fold, whose ranking is substrate dependent. Strikingly, 10, previously Behçet’s disease, consistently low-activity outlier, suggesting significant percentage carry subactive gene. Enzymatic analysis revealed differ both catalytic efficiency affinity, differences change intermediate accumulation multistep trimming reactions. Alterations efficacy an allosteric inhibitor targets regulatory site suggest allotypic influences communication active site. Our work defines landscape activity populations demonstrates how induce substrate-dependent antigen processing, thus contributing, synergy haplotypes, response chronic conditions. Major (MHC) molecules (human leukocyte antigens [HLAs] humans) are polymorphic genes tens thousands different allomorphs identified date (1Robinson J. Barker D.J. Georgiou X. Cooper M.A. Flicek P. Marsh S.G.E. IPD-IMGT/HLA database.Nucleic Acids Res. 2020; 48: D948-D955PubMed Google Scholar). MHC (MHCI) bind small protein fragments (peptides) originate from normal cellular or pathogen then translocate cell surface present cargo cytotoxic T-lymphocytes (2Rock K.L. Goldberg A.L. Degradation generation I-presented peptides.Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (771) MHCI predominantly affects structure binding groove allows large variety peptide sequences. MHCIs endoplasmic (ER) assistance peptide-loading (3Blees A. Januliene D. Hofmann T. Koller N. Schmidt C. Trowitzsch S. Moeller Tampe R. Structure MHC-I complex.Nature. 2017; 551: 525-528Crossref (150) While tend 8 11 amino acids long (the majority which 9mers), many enter ER be substantially longer (4Cascio Hilton Kisselev A.F. Rock 26S proteasomes immunoproteasomes produce mainly N-extended versions peptide.EMBO 2001; 20: 2357-2366Crossref (254) Two ER-resident aminopeptidases, 2 (ERAP1 ERAP2), catalytically process precursor define pool available onto (5Weimershaus M. Evnouchidou I. Saveanu L. van Endert Peptidases ligands.Curr. Opin. 2013; 25: 90-96Crossref (50) The gene also polymorphic, coding confer susceptibility notably conditions, often epistasis HLA alleles, emphasize critical role (6Strange Capon F. Spencer C.C. 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Rheumatol. 27: 349-356Crossref genetic diseases, such HLA-B27-associated (AS), HLA-B51-associated Behçet's HLA-A29-associated birdshot uveitis, led hypothesis conditions driven changes direct result alterations preferences (10Kuiper J.J.W. Setten J.V. Devall Cretu-Stancu Hiddingh Ophoff R.A. Missotten Velthoven M.V. Den Hollander A.I. Hoyng C.B. James Reeves Cordero-Coma Fonollosa Adan al.Functionally distinct hallmark HLA-A29-(birdshot) uveitis.Hum. Mol. 2018; 4333-4343PubMed 11Chen Fischer Peng Y. McHugh K. Ternette Hanke Dong Elliott Shastri Kollnberger Kessler B. Bowness Critical determining length sequence bound presented HLA-B27.Arthritis 2014; 66: 284-294Crossref (62) 12Guasp Gomez-Molina Marcilla Barnea Admon Lopez peptidome Behcet's disease-associated HLA-B?51:01 includes two subpeptidomes differentially shaped 1.Arthritis 68: 505-515Crossref (42) 13Sanz-Bravo Campos Mazariegos M.S. Dominant polymorphism R528K shaping HLA-B27 through differential processing determined multiple residues.Arthritis 67: 692-701Crossref (34) Several described function enzyme (14Evnouchidou Kamal R.P. Seregin S.S. Goto Tsujimoto Hattori Voulgari P.V. Drosos A.A. Amalfitano York I.A. Stratikos single nucleotide polymorphisms influencing basic enzyme.J. 2011; 186: 1909-1913Crossref (103) 15Reeves Colebatch-Bourn Edwards C.J. Functionally distinguish Ankylosing Spondylitis.Proc. Natl. Acad. Sci. U. 111: 17594-17599Crossref (71) Mechanisms proposed underlie this effect include interactions (16Giastas Mpakali Papakyriakou Lelis Kokkala Neu Rowland Liddle Georgiadis Mechanism selection 1.Proc. 2019; 52: 26709-26716Crossref (18) Scholar), on conformational dynamics (17Papakyriakou intracellular aminopeptidases.Front. 8: 946Crossref (20) expression level (18Hanson Cuddihy Haynes Loo Morton Oppermann Thomas G.P. Le Cao K.A. Kenna T.J. Brown Genetic variants immune-mediated diseases influence isoform profile.Arthritis 70: 255-265Crossref (27) 19Costantino Talpin Kadi Leboime Said-Nahal Bonilla Letourneur Leturcq Ka Z. Breban influenced nonsynonymous spondyloarthritis.Arthritis 1525-1534Crossref (41) However, all possible nine (i.e., allotypes) (20Goto Ishii Reduced hypertension-associated Lys528Arg mutant adipocyte-derived leucine (A-LAP)/ER-aminopeptidase-1.FEBS Lett. 2006; 580: 1833-1838Crossref (89) Scholar) occur at equal population (9Ombrello Rather, encode limited palette maintained high (>1%) suggests asymmetry allotypes. This supported fact some protective, whereas others risk (21Alvarez-Navarro structure, pathogenetic other MHC-associated diseases.Mol. 57: 12-21Crossref (58) A deep understanding rather than unraveling physiological impact disease. Previous studies several Most defined combination acid positions 56, 127, 276, 346, 349, 528, 575, 725, 730. Ombrello al. reported 10 three European East Asian Ancestry (n = 160). additional discovered patient cohorts AS 72) (15Reeves oropharyngeal squamous carcinoma 25) (22Reeves Wood Ottensmeier C.H. King E.V. G.J. HPV epitope correlate extent CD8(+) T-cell tumor infiltration OPSCC.Cancer 7: 1202-1213Crossref (13) These however controversial rare (23Wordsworth B.P. Vecellio Roberts A.R. Reply al.: No evidence haplotypes haplotype spondylitis.Proc. 114E1577Crossref (1) 24Roberts Appleton L.H. Cortes Lau Watts Wordsworth attributable genotypes combinations.Proc. 114: 558-561Crossref (21) contributed our emerging controversy remains common, systematic lacking. 1000 Genomes Project (25Genomes Auton Brooks L.D. Durbin R.M. Garrison E.P. Kang H.M. Korbel J.O. Marchini J.L. McCarthy McVean G.A. Abecasis G.R. global reference variation.Nature. 526: 68-74Crossref (6822) populations. comprehensively because affinity. findings potential strongly synergize epitope-dependent manner, enhance natural Analysis Genome Aggregation Database contains 125,748 exome sequences (26Karczewski K.J. Francioli L.C. Tiao Cummings B.B. Alfoldi Wang Q. Collins R.L. Laricchia K.M. Ganna Birnbaum D.P. Gauthier Brand H. Solomonson N.A. Rhodes al.The mutational constraint spectrum quantified 141,456 humans.Nature. 581: 434-443Crossref (1615) using 1% cutoff qualify genomic missense variant only namely 12, 730, consistent previous Since position 12 lies signal normally excised after translocation into does appear mature protein, we focused remaining positions. could theoretically organized 29 discreet To exploited ordered along one chromosome) 5q15 phase 3 (27Song Li W. Xie Hou You Cytokine storm induced SARS-CoV-2.Clin. Chim. Acta. 509: 280-287Crossref (203) Table S1. Correlations indicate linkage disequilibrium S2 generally 1. An analogous UK biobank similar results (Table S3) (28Sudlow Gallacher Beral V. Burton Danesh Downey Green Landray Liu Matthews Ong Pell Silman al.UK biobank: open access resource identifying causes middle old age.PLoS Med. 12e1001779Crossref (2262) although constitute 99.9% population, before. Overall, were able identify least six frequencies over 0.5% 1; 11–16). confirmed larger setting there Regardless, given extensive use literature near-complete coverage (>94% globally, >99.9% population), proceeded characterization copies gene, analyzed samples (Fig. 1A Tables S4 S5). was followed 2-2 homozygous, cumulatively account almost 20% population. Interestingly, 8-2 found about twice frequent (11%) predicted random distribution (25.6% × 21.8% 5.6%). addition prevalence homologous ERAP2, rs2549782 rs2248374 (29Evnouchidou Birtley Zervoudi Samiotaki Panayotou Giastas Petrakis Saridakis Mavridis I.M. induces specificity switch leads altered processing.J. 2012; 189: 2383-2392Crossref 30Andres A.M. Dennis M.Y. Kretzschmar W.W. Cannons Lee-Lin S.Q. Hurle Program N.C.S. Schwartzberg P.L. Williamson S.H. Bustamante C.D. Nielsen Clark A.G. E.D. Balancing maintains form undergoes nonsense-mediated decay presentation.PLoS 6e1001157Crossref (156) Of four combinations, detect sample, [G,A] (defined (30Andres Scholar)) 44.7% [T,G] B 55.3% samples. [G] allele no detectable 31.3% database should therefore expression. co-occurrence dependent background Dutch Spanish controls; therefore, interested see if set. calculated each group carrying particular S6), interesting correlations. [8,8] 19.2% [A,A] allotype, much more compared whole (5.8%). In addition, [2,2] 22.3% [B,B] (8.2% population). correlation specific may functionally relevant discussed later article.Table 1List populationsAllotypeAmino composition SNPPopulation (%)56127276346349528575725730ALLAFRAMREUREASSAS1EPIGMKDRQ6.10.512.114.00.07.62ERIGMKDRQ25.628.122.614.442.618.43ERIGMKDRE8.511.45.86.62.714.54ERIGMRDRE3.23.08.91.02.42.45ERIDMRDRE6.28.43.96.30.010.96EPIGMRDRE5.84.010.29.20.76.57KPIGMRDRE7.22.98.54.117.15.08EPMGMRDRE21.817.414.021.528.526.79EPMGMRNRE0.10.00.40.40.00.010EPIGVRNQE9.63.512.422.45.86.5Subtotal94.179.298.899.999.898.511EPIDMRDRE2.79.90.70.10.00.012KPIDMRDRE1.45.20.10.00.00.013EPIGVRNRE0.83.00.10.00.00.014EPMGMRNQE0.41.70.00.00.00.015ERMGMRDRE0.20.50.00.00.10.116EPMGMRDRQ0.120.00.00.00.00.6Subtotal5.620.30.90.10.10.7Total99.799.599.710099.999.2AFR, African; ALL, cumulatively; AMR, American; EAS, Asian; EUR, European; SAS, South Asian.Bold indicates membership ALL Italics subtotals totals. Open table tab AFR, Asian. Bold characterize listed scattered throughout ERAP1, away center S1) categorized groups: (a) lie outside central cavity accommodates S1A) (b) line interior make S1B). first well-established dipeptide substrates. hydrolysis l-leucine-7-amido-4-methylcoumarin (Leu-AMC) Figure 1B. There approximately twofold spread activities 9, 10-fold less 1B Fig. S2). relationship concentration linear 150 ?M substrate, allowed calculation kcat/KM ratio Leu-AMC 1C S7). Allotype all, 18-fold active. obtain full Michaelis–Menten (MM) analysis, employed chromogenic l-leucine-para-nitroanilide (Leu-pNA) (31Nguyen T.T. Chang S.C. Zikos Stern L.J. Structural basis Struct. Biol. 18: 604-613Crossref (138) Data fit best MM model demonstrated 32Giastas High-resolution crystal phosphinic transition-state analogue inhibitor.ACS Chem. 10: 708-713Crossref (16) allowing us calculate parameters 1D affinity (Khalf) maximal (Vmax). Similar Leu-AMC, hydrolyzing Leu-pNA, unfortunately precluded reliable Khalf Vmax allotype. trims N-terminally extended precursors peptides, turned physiologically peptides. Recently cocrystal structures 15mer 10mer analogs processed internal cavity, while making residues selectivity measured rate N-terminus backbone cocrystallized analogs, LLRIQRGPGRAFVTI LLKHHAFSFK 2, B). Like substrates, recorded rates. Although again efficient, pronounced smaller Notably, efficient 9 15mer. pattern significantly different. Allotypes 4 6 trimmed fastest, 5 7 fastest. peptide–enzyme 33Chang Momburg Bhutani aminopeptidase, lengths “molecular ruler” mechanism.Proc. 2005; 102: 17107-17112Crossref (223) better understand mechanism behind rates, utilized recently developed assay suitable follows 9mer YTAFTIPSI 2C) (34Liddle Hutchinson Kitchen Cecconie Holmes D.S. Jones Korczynska Koumantou Lea J.D. Nickels Pemberton Phillipou Schneck al.Targeting discovery product modulator presentation.J. 63: 3348-3358Crossref