JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001076/files/111_Maruhashi_3_V20A_M2_IP_QEp.rawhttps://storage.jpostdb.org/JPST001076/files/113_Maruhashi_4_L14Q_V20A_M2_IP_QEp.rawhttps://storage.jpostdb.org/JPST001076/files/109_Maruhashi_2_WT_M2_IP_QEp.rawhttps://storage.jpostdb.org/JPST001076/files/200901_Maruhashi_medium_M2_IP_QEp.pdResultprimaryOK200ProteomicsHidetaka KosakoHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST001076Tokushima UniversityjPOSTLymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.Maruhashi Takumi T, Sugiura Daisuke D, Okazaki Il-Mi IM, Shimizu Kenji K, Maeda Takeo K TK, Ikubo Jun J, Yoshikawa Harunori H, Maenaka Katsumi K, Ishimaru Naozumi N, Kosako Hidetaka H, Takemoto Tatsuya T, Okazaki Taku TModb1, PP17, Ifm(2)RU1, Dm II, MHC, Activity, DnMod, Laboratory, Blast, PP19, Stp, Mus domesticus, Lymphoblast Transformation, Autoimmunities, Bsh, CASP-14, protein, Tumor, gene5, House Mouse, QA1, peptide, Myo, dmTAF[[II]]230, Polypeptides, B37, peptido, mhc, diseases, fibrinogen gamma chain, ifm(2)2, Alleviating interaction, diseases and disorders, autoimmunity, 1110025J15Rik, protein aggregate, Leprb, DmelCG4190, HSP67, Hspg3, Autoimmune condition, C1orf215, FBgn0002741, DM - Diabetes mellitus, CG17927, LFIRE1, Autoimmune Response, fibrinogen, Immune Processes, Immune Responses, human disease, Man (Taxonomy), peptides, mMHC, TFIID TAF250, Autoimmune disease, Myo2, cel, l(2)M66, LFIRE-1, ataxia, DROMHC, Swiss Mice, DmelCG7693, 4624, Pp17, Pp18, NMMII, gene3, ifm(2)RU2, Diabetes mellitus, Immune, malignant neoplasm, gene 3, Pp19, NMM, dentatorubral pallidoluysian atrophy, HLA-6.2, EA1.2, Homo sapiens disease, Malignancies, P18, l(3)07551, l(2)k10423, P19, Mhc36B, Myo II, l(2)36Ae, db, Tumors, sMHC, and dementia, Ligand, dTAF[[II]]230, Coagulation, HRS, DM, Process, Modern, CASP14, mouse, TAF200, Mhc-c[1], RCB2758, ifm(2)RU1, TAFII-250, seizures, TAF250/230, myoII, results, Sht, OB-RGRP, NOD, Nod, TAFII250, fibrinogen alpha chain, Benign, DmNod, KIF22, Mini-ICE, LAP18, Diseases, EA2.1, NOS, hsp67B, Lap18, dOSR1, Mus musculus, Lymphocyte Stimulation, Stimulation, Caspase-14 subunit p10, CG7693, mice, Swiss Mouse, Benign Neoplasms, Caspase-14 subunit p19, small hsp locus 67B, Blood Coagulation Factor I, CG17603, MICE, TAF[[II]], human, Obr, domesticus, Diabetes NOS, Malignant Neoplasms, loss of, gamma-Fibrinogen, disease, Taf250, SR3-5, CG4190, medical condition., Mouse, Polypeptide, MyoII, obl, TAF230, gene-3, Zip, ZIP, Autoimmune Responses, 3.4.22.-, other disease, d230, human being, Blastogenesis, Neoplasms, Blast Transformation, Benign Neoplasm, Gene, dTAFII250, mini-ICE, Myo-II HC, l(1)TW6, protein-containing complex, EfW1, Malignant, Human, DmelCG1763, Dm nmII, Homo sapiens, dmTAF1, House, Taf230, Gene Products, Diabetes mellitus (disorder), HP-041, Mus musculus domesticus, disease or disorder, gamma Fibrinogen, SMN, Smn, Mice, Man, CG15792, TAF250, D12S755E, 19k, Taf200, Naito Oyanagi disease, dTAF[[II]]250, Activation, MRP, Mrp, Malignancy, Myo-II, Ifm(2)2, cell, Swiss, dSTRAD, ligand, zip/MyoII, DmMHC, obese-like, Taf1p, Coagulation Factor I, suppressive genetic interaction (sensu inequality), DmelCG17927, non-neoplastic, Neoplasias, dTAF250, Immune Response, Nup, Pig, disorder, Lag, LAG, peptidos, DmnmII, TAF, chorea, Immune Process, Diabetes, Lymphocyte, diabetes, l(2)02957, Cancer, Autoimmunity, Transformation, Autoimmune disorder, TAF[[II]]250, HSPB8, Malignant Neoplasm, Lymphoblast, protein complex, Factor I, DroMII, HFREP1, Proteins, Hsp-G3, disorders, lag, DRPLA, CG1763, l(3)84Ab, E(br), BG:DS00004.13, Peptide, Cell, dTAF230, diabetes mellitus (disease), fibrinogen beta chain, polypeptide, Hsp67BC, MT, native protein, nmMHC, Fibrinogens, Mus, p230, Protein, Neoplasm, TAF[[II]]250/230, TFIID, condition, Mhc-c, STE20-like/SPAK, myoclonic epilepsy with choreoathetosis, DmelCG15792, Taf[[II]]250, LEPROT, Pr22, primary cancer, TAF[[II]]230, distinct, diabetes mellitus, Op18, Non-obese Diabetic, House Mice, TAF[II]250, Cancers, malignant tumor, Laboratory Mice, Protein Gene Products, Gene Proteins, MyHC, HSP67Bc, DmelCG17603, PR22, OP18, Modern Man, Response, Dronm-MII, anon-WO0140519.37, Lymphocyte Transformation, Peptid, General activity, Laboratory Mouse, Neoplasia, zpr, Dm-HSP67Bc, nmy-2, TAF1liquid chromatography tandem mass spectroscopy, LC-MS2, PP17, determination, PP19, Proteins, LC-MS/MS, lag, Gene, RCB2758, LC-MS-MS, Mutations, Avazyme, LC/MS/MS, Protein, LC-MSMS, LAP18, Gene Products, SMN, Smn, 1110025J15Rik, STE20-like/SPAK, C1orf215, Lap18, dOSR1, 19k, Pr22, LCMSMS, CG7693, Op18, dSTRAD, Alpha-Chymotrypsin Choay, chemical analysis., DmelCG7693, Alphacutanée, 4624, Protein Gene Products, Pp17, Pp18, Gene Proteins, PR22, liquid chromatography-tandem mass spectroscopy, OP18, Pp19, Pig, liquid chromatography tandem mass spectrometry, Lag, LAG, assay, P18, l(3)07551, P19liquid chromatography tandem mass spectroscopy, l(1)d norm-12, PP17, Bru, BG:DS00941.4, EG:17A9.1, Raw, CG7793, determination, BR_C, PP19, Aminosaeure, 2Bc, l(2)Sos, protein, BR-C, nodus primitivus., Alkylations, Mutations, Polypeptides, Background, L-Isomer Methionine, Methionine, peptido, l(1)2Bad, A18, br-C, l(1)2Bab, dorsal marginal zone, T-PA, br-Z1, Cultural, br-Z3, PP1, PP2, 1110025J15Rik, protein aggregate, BR-c, Br-C, l(1)dn1, E(sev)2A, WMS, C1orf215, Svc, Br-Z4, rbp, AU020998, ethnicity, LCMSMS, amino acids, peptides, l(1)G0318, de12, E(var)189, M, BR, l(1)n34, BrC-Z1, DmelCG7693, 4624, Br-C-Z3, Pp17, Pp18, HUMRPL29, ppm, Pp19, Br, BRC-Z2, BRC-Z3, BRC-Z1, P18, BRC-Z4, Half Cystine, l(3)07551, P19, GPHYSD2, Racemethionine, CG11491, SGS, rdp, LC-MS2, rds, Zinc Cysteinate, Aminokarbonsaeure, BR-C Z1, l(1)d.norm.1, acetylation, LC-MS/MS, 2-Amino-4-(methylthio)butyric acid, RCB2758, L27', Search, l(1)npr-1, DmelCG7793, uq, predicted, l(1)npr1, ACMICD, Avazyme, Cultural Background, soluble, Cultures, LAP18, dme-SOS, Henson's node, Methionin, nrp1, Hmet, br24, dSos, br25, Lap18, parent ion, dOSR1, Amino Acid, Acid, D8Ertd2e, Cultural Beliefs, CG7693, 34Ea, RPL29_3_370, npr-1, L-Cysteine, MASS, nanospray, precursor ion, liquid chromatography-tandem mass spectroscopy, Biocatalyst, DMZ, npr, l(1)pp-2, l(1)pp-1, L27A, Acids, Polypeptide, Liquimeth, AW212668, Cysteine Hydrochloride, 2-amino-4-(methylsulfanyl)butanoic acid, Biocatalysts, l(1)G0018, DmelCG11491, Aminocarbonsaeure, L Isomer, l(1)ts144, FBN, Gene, l(1)2Ba, l(1)2Bb, Z1, l(1)2Bc, Z2, EK2-8, precursor, Z3, Z4, protein-containing complex, LC-MS-MS, CG11511, RPL29P10, EG:25D2.1, CG11514, ECTOL1, nrp, LC-MSMS, Br-C Z2, Gene Products, l(1)ts132, L-Methionine, l(1)ts376, SMN, Smn, Del(8)44H, Half-Cystine, PATISS, l(2)br25, Pedameth, Backgrounds, l(2)br24, l(1)ESHS5, 19k, Search Engines, TPA, dSTRAD, L Cysteine, nprl, l(2)k06321, Acetylations, Cultural Relativisms, OCTD, tPA, l(1)G0042, l(1)G0284, Enzyme, 2-amino-4-(methylthio)butanoic acid, l(1)2Bd, 10^[-6], stem node, HIP, Pig, Customs, npr1, Lag, LAG, peptidos, l(1)PP1, l(1)pp1, l(2)34Ea, culture, SOS, l(1)pp2, 2.3, metionina, EG:123F11.1, DL-Methionine, protein complex, Proteins, L29, lag, ecs, BrZ3, sos, Cultural Backgrounds, l(1)G0284a, L-Isomer, Cell, Peptide, Engine, LC/MS/MS, native protein, o.c.c., l(2)k05224, Protein, chemical analysis, MFS1, STE20-like/SPAK, Data Base, Sos1, WMS2, Met, Pr22, Col4a-1, nodal stem, Op18, l(1)ts358, EY2-3, Magnetic, Alpha-Chymotrypsin Choay, Episome, Amino, Alphacutanée, Protein Gene Products, Episomes, Gene Proteins, Su(tor)2-2, 2B5, PR22, l(1)G0401, OP18, SSKS, Plasmid, alpha-amino-gamma-methylmercaptobutyric acid, Relativisms, liquid chromatography tandem mass spectrometry, BRC, Peptid, assay, Relativism, variable, Cultural RelativismZip, ZIP, Autoimmune Responses, PP17, Ifm(2)RU1, Dm II, MHC, PP19, Stp, Neoplasms, Autoimmunities, Benign Neoplasm, Myo-II HC, Bsh, Tumor, Malignant, QA1, peptide, Myo, Polypeptides, peptido, Dm nmII, mhc, ifm(2)2, autoimmunity, SMN, Smn, 1110025J15Rik, Immune., CG15792, Autoimmune condition, C1orf215, FBgn0002741, CG17927, 19k, Autoimmune Response, Immune Processes, Immune Responses, peptides, mMHC, Autoimmune disease, MRP, Mrp, Malignancy, Myo-II, Myo2, Ifm(2)2, dSTRAD, l(2)M66, ligand, zip/MyoII, DmMHC, DROMHC, DmelCG7693, 4624, DmelCG17927, Pp17, Pp18, NMMII, Neoplasias, ifm(2)RU2, Immune Response, Immune, Nup, malignant neoplasm, Pp19, Pig, NMM, HLA-6.2, Lag, LAG, peptidos, EA1.2, Malignancies, DmnmII, P18, l(3)07551, l(2)k10423, P19, Immune Process, Mhc36B, Myo II, l(2)02957, l(2)36Ae, Cancer, Tumors, sMHC, Autoimmunity, Autoimmune disorder, Malignant Neoplasm, Process, DroMII, lag, Mhc-c[1], RCB2758, E(br), ifm(2)RU1, myoII, Peptide, Sht, polypeptide, MT, Benign, nmMHC, LAP18, Neoplasm, Mhc-c, EA2.1, STE20-like/SPAK, Lap18, DmelCG15792, dOSR1, Pr22, primary cancer, CG7693, Op18, Benign Neoplasms, Cancers, malignant tumor, Malignant Neoplasms, MyHC, PR22, OP18, Response, Dronm-MII, anon-WO0140519.37, Peptid, Polypeptide, MyoII, Neoplasia, zpr, nmy-2falseMature proteins of LAG-3 with or without mutations were digested with chymotrypsin followed by data-dependent LC-MS/MS analysisThe FLAG-tagged soluble monomeric proteins of LAG-3-WT, LAG-3-V20A, and LAG-3-L14Q/V20A were immunoprecipitated from culture supernatants of Plat-E cells transfected with the corresponding plasmid vectors using ANTI-FLAG M2 Magnetic Beads. Proteins on the beads were digested with chymotrypsin (Roche) followed by reduction and alkylation. The peptides were subjected to data-dependent LC-MS/MS analysis using an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Raw data were directly analyzed against the SwissProt database restricted to H. sapiens supplemented with predicted amino acid sequences of LAG-3 with or without mutations starting from A18 to L29 using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) with Sequest HT search engine. The search parameters were as follows: (a) chymotrypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the percolator node.Mon Apr 11 00:00:00 BST 2022PXD023929960635413245