Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of

This information is current as Edward J. Burgess, Laura R. Hoyt, Matthew J. Randall, of September 24, 2021. Madeleine M. Mank, Joseph J. Bivona III, Philip L. Eisenhauer, Jason W. Botten, Bryan A. Ballif, Ying-Wai Lam, Matthew J. Wargo, Jonathan E. Boyson, Jennifer L. Ather and Matthew E. Poynter

J Immunol 2018; 201:2377-2384; Prepublished online 29 Downloaded from August 2018; doi: 10.4049/jimmunol.1800503 http://www.jimmunol.org/content/201/8/2377 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/08/28/jimmunol.180050 Material 3.DCSupplemental References This article cites 48 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/201/8/2377.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A

Edward J. Burgess,*,†,‡ Laura R. Hoyt,*,‡ Matthew J. Randall,*,‡ Madeleine M. Mank,*,‡ Joseph J. Bivona, III,*,†,‡ Philip L. Eisenhauer,x Jason W. Botten,*,†,x,{ Bryan A. Ballif,‖ Ying-Wai Lam,‖ Matthew J. Wargo,*,†,{ Jonathan E. Boyson,*,†,# Jennifer L. Ather,*,‡ and Matthew E. Poynter*,†,‡

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these . In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory

cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli– Downloaded from derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with or addition of a lipopeptide to eukaryotic cell–derived hSAA1 inhibited or induced the production of TNF-a from , respectively. Our results suggest that a function of SAA is in the binding of TLR2- stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant derived from eukaryotic cells. The Journal of Immunology, 2018, 201: 2377–2384. http://www.jimmunol.org/ he serum amyloid A (SAA) family of acute-phase proteins SAA1 and SAA2 are highly homologous and predominantly produced have been studied for decades as robust biomarkers for a by the liver, whereas SAA3 is an acutely expressed isoform produced T wide array of inflammatory and autoimmune disorders as in nonprimate mammals (2), and the constitutively expressed SAA4 well as for their contribution to AA amyloidosis. Increasing ∼1000- does not increase in response to infection or injury (3). fold in the serum in response to infection and injury, SAA proteins Given its rapid and robust increase under inflammatory and are evolutionarily conserved and are the major acute-phase pro- autoimmune conditions, it has long been speculated that SAA is a teins in vertebrates (1). Multiple isoforms of SAA are expressed in mediator of the inflammatory process. Specifically, we and others the liver (the largest source of acute-phase reactants) as well as in have reported that the proinflammatory effects of SAA are largely hematopoietic and nonhematopoietic cells throughout the body. mediated through the activation of TLR2 (4–18). However, the by guest on September 24, 2021 Escherichia coli–derived recombinant form of human SAA that is almost uniformly used by investigators (the form of *Vermont Lung Center, University of Vermont, Burlington, VT 05405; †Cellular, Molecular, and Biomedical Sciences Program, University of Vermont, human SAA1 [apoSAA], a lipid-binding apolipoprotein that is a Burlington, VT 05405; ‡Division of Pulmonary Disease and Critical Care, Depart- x constituent of plasma lipoprotein) elicits strong proinflammatory ment of Medicine, University of Vermont, Burlington, VT 05405; Immunobiology responses not shared with the endogenous form of SAA. Specif- Division, Department of Medicine, University of Vermont, Burlington, VT 05405; {Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, ically, although recombinant apoSAA promotes neutrophil acti- VT 05405; ‖Department of Biology, University of Vermont, Burlington, VT 05405; # vation and proinflammatory cytokine production, human plasma and Department of Surgery, University of Vermont, Burlington, VT 05405 containing highly elevated levels of SAA displays neither of these ORCIDs: 0000-0002-0350-4154 (L.R.H.); 0000-0003-3840-8952 (M.J.R.); 0000- effects (19, 20). Furthermore, transgenic overexpression of mouse 0002-0617-4736 (M.M.M.); 0000-0001-5659-749X (M.J.W.); 0000-0003-2673- 9148 (J.E.B.). (21) or human (20, 22) SAA1 in mice to levels that recapitulate Received for publication April 11, 2018. Accepted for publication August 4, 2018. those in the circulation of humans does not elicit a proinflammatory This work was supported by National Institutes of Health Grants R01 HL107291, state. These results imply that further characterization of the dif- R01 HL133920 (both to M.E.P.), R01 AI103003 (to M.J.W.), T32 HL076122 (to E.J.B. ferences in proinflammatory activities between recombinant and and M.J.R.), P30 GM103532, P20 GM103496, and P20 GM103449. Research reported endogenous SAA is warranted. in this publication was supported by an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health SAA proteins associate with high-density lipoprotein (HDL), under Grant P20 GM103449. The contents of this work are solely the responsibility displacing human (apoA1) as the predominant of the authors and do not necessarily represent the official views of the National Institute apolipoprotein during inflammation (1, 23). Interestingly, the ca- of General Medical Sciences or the National Institutes of Health. pacity of SAA to stimulate cells via TLR2 has been reported to be Address correspondence and reprint requests to Dr. Matthew E. Poynter, University of Vermont, Department of Medicine, Division of Pulmonary and Critical Care, 89 only when it is not incorporated into HDL (21). SAA has been Beaumont Avenue, Given E410A, Burlington, VT 05405. E-mail address: matthew. shown to prevent the entry of enveloped viruses into cells (24–26) [email protected] and reported to opsonize Gram-negative bacteria (27, 28). The The online version of this article contains supplemental material. outer membrane protein A (OmpA) of E. coli has been reported to Abbreviations used in this article: apoA1, human apolipoprotein A1; apoSAA, the bind SAA1, enabling it to augment the ability of neutrophils to apolipoprotein form of human SAA1; DC, dendritic cell; FPLC, fast protein liquid chromatography; HDL, high-density lipoprotein; HEK, human embryonic kidney; ingest invading bacteria (27). Furthermore, SAA functions as a hSAA1, recombinant human SAA1; Kav, elution volume parameter; LPL, lipoprotein circulating chaperone for the small, lipophilic vitamin A deriva- lipase; LSM, lymphocyte separation medium; OmpA, outer membrane protein A; tive retinoic acid (29) and can associate with phospholipids to form PEI, polyethylenimine; SAA, serum amyloid A; V , void volume. o larger particles (30). As SAA clearly has the capacity to associate Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 with lipid-rich particles and compounds, we hypothesized that other www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800503 2378 EUKARYOTE-DERIVED SAA IS NOT PROINFLAMMATORY lipophilic molecules, such as bacterial lipopeptides, may also as- new tubes, and frozen at 220˚C until analysis. For transfection, 5.6 3 104 sociate with SAA. Bacterial lipopeptides are potent activators of HEK293T cells were seeded per square centimeter in each well of 6- or 96- TLR2, whereby they induce the production of proinflammatory well plates (for expression of SAA or TLR1/2, respectively) and transfected 1 d later with 11.1 ml/cm2 DMEM containing 0.2 mg/cm2 plasmid and cytokines and other effects that have been attributed to recombinant 0.9 mg/cm2 of PEI per well. forms of SAA produced in E. coli. The objective of our studies was to compare the effects of E. coli–derived and eukaryotic cell– Cytokine and SAA quantitation derived SAA1 proteins on TNF-a production from macrophages, Cell supernatants were analyzed for mouse TNF-a, IL-1b, or IL-4 using the stimulation of TLR2, and the induction of Th17 responses, as ELISA kits from BD Biosciences. Human IL-8, IL-6, TNF-a, IL-1b, and well as to examine the contribution of bacterial lipoproteins to the SAA1 as well as mouse IL-5, IL-13, IL-17A, and IFN-g were measured by capacity to induce the aforementioned proinflammatory effects. ELISA using DuoSet reagents from R&D Systems. All ELISAs were performed according to manufacturer’s instructions.

Materials and Methods Fast protein liquid chromatography Reagents Two hundred and fifty micrograms of apoSAA was run on a Superdex 75 size-exclusion column (GE Healthcare Life Sciences, Pittsburgh, PA) in Chemicals were from Thermo Fisher Scientific (Hampton, NH) unless PBS (pH 7.2) running buffer, quantitated for total protein (OD280)asit noted otherwise. Recombinant human apoSAA, recombinant human SAA1 eluted from the column (90 fractions of ∼330 ml each were collected), and (hSAA1), and apoA1, all made in E. coli, were from PeproTech (Rocky compared with the elution time of protein molecular mass standards to Hill, NJ). Recombinant mouse SAA1 made in E. coli was from R&D estimate its size. Briefly, a standard curve was generated by plotting the Systems (Minneapolis, MN). hSAA1 made in human embryonic kidney elution volume parameter (K ) of several standards purchased from GE av Downloaded from (HEK) cells was from OriGene (Rockville, MD). Ultrapure E. coli O111: Healthcare (RNase A, aprotinin, carbonic anhydrase, OVA, and con- B4 LPS and Pam3CSK4 were from InvivoGen (San Diego, CA). Plasmid albumin) versus their log molecular masses. The K of SAA was read pcDNA3 was from Invitrogen (Carlsbad, CA). pcDNA3.1+ encoding hu- av from this curve to estimate its molecular mass (∼76 kDa). Kav was cal- man SAA1 was from GenScript (Piscataway, NJ). Plasmid pCMV6-XL5 culated as follows: blue dextran was used to calculate the void volume (V ) encoding human SAA1 was from OriGene. E. coli transformed with o of the column, and the total bed volume (Vt) was 24 ml. Kav = (Elution pLX304 plasmids encoding human TLR1 and TLR2 were from the DNASU volume (V ) 2 V )/(V 2 V ). Plasmid Repository (Tempe, AZ). Plasmids from these bacteria were pu- e o t o

rified using EndoFree Plasmid Kits from Qiagen (Germantown, MD). Mass spectrometry analysis http://www.jimmunol.org/ Polyethylenimine (PEI) was from Polysciences (Warrington, PA). Mini- PROTEAN TGX precast 4–20% polyacrylamide gels were from Bio-Rad For in-gel digestion and subsequent analyses, apoSAA (50 mg) in Laemmli (Hercules, CA). Immobilon nitrocellulose transfer membranes and Amicon buffer with 5% 2-ME was separated on a 4–20% Tris-glycine gel at 140 V Ultra centrifugal filter units with a 3-kDa molecular mass cutoff were from for 1 h 15 min. The gel was then stained in 30% Coomassie Brilliant Blue EMD Millipore (Billerica, MA). Lipoprotein lipase (LPL) from Pseudo- (40% methanol, 20% acetic acid, and 0.1% Brilliant Blue R [Sigma- monas sp. and OVA were from Sigma-Aldrich (St. Louis, MO). TOP10 Aldrich]) (diluted in destain) overnight, destained in 30% methanol and chemically competent E. coli were from Invitrogen. Anti-CD3 and anti- 20% acetic acid, and imaged using a CanoScan 8800F scanner (Canon, CD28 Abs were from BD Biosciences (San Jose, CA). Melville, NY), and a cut map was created to divide the lanes into 12 specific cuts. Proteins within the gel slices were reduced in 25 mM DTT Cells for 30 mins, alkylated in-gel in 10 mM iodoacetamide for 45 mins, and subjected to two rounds of dehydration with acetonitrile and rehydration by guest on September 24, 2021 Peripheral blood was collected from normal adult human donors under the with water prior to a final dehydration in acetonitrile. To the dry gel slices, approval of the University of Vermont Institutional Review Board (protocol 25 ml of 12 ng/ml sequence-grade trypsin (Promega, Madison, WI) in no. M10-171). For the preparation of PBMCs, blood collected into EDTA- 50 mM ammonium bicarbonate was added. The samples were placed on coated tubes was diluted 1:1 in HBSS (without calcium or magnesium), ice for 30 min and digested at 37˚C overnight. Tryptic peptides were layered over lymphocyte separation medium (LSM; MP Biomedicals, Santa extracted with 2.5% formic acid in 50% acetonitrile while spinning in a Ana, CA), and centrifuged at 500 3 g for 30 min at room temperature. The microcentrifuge at 13,000 rpm for 10 min. The supernatant was collected, top layer of plasma was aspirated and discarded, leaving 2–3 mm above and the gel slices were dehydrated by twice incubating with 100% ace- the buffy coat, and the buffy coat and half the lower LSM layer were tonitrile and collecting all the extractions from a given gel slice in the same aspirated, mixed with HBSS, and washed twice. Neutrophils were enriched tube, and solvent was removed using a vacuum centrifuge at 37˚C. The by resuspending the pellet obtained during the LSM preparation of PBMCs peptides were resuspended in 2.5% acetonitrile and 2.5% formic acid, in 20 ml of HBSS and adding 20 ml of 3% dextran in HBSS, which was loaded using a Micro AS autosampler (Thermo Fisher Scientific, Pitts- subsequently mixed by inversion several times. Erythrocytes were allowed burgh, PA), and separated in a microcapillary column packed with 12 cm to settle for 20 min at room temperature, after which the neutrophil-rich of Magic C18 200-A5-˚ mm material (Michrom Bioresources, Auburn, supernatant was transferred to a new tube and washed with HBSS. CA). The peptides were separated and eluted with a 5–35% acetonitrile Erythrocytes were lysed by two rounds of resuspending the pellet in 10 ml (0.15% formic acid) gradient using a Surveyor Plus HPLC pump instru- of hypotonic lysis buffer for 30 s followed by the addition of an equal ment (Thermo Fisher Scientific) over 40 min, after a 15-min isocratic volume of re-equilibration buffer and washing. Splenocytes and bone loading at 2.5% acetonitrile and 0.15% formic acid. Mass spectra were marrow were collected from C57BL/6J mice purchased from The Jackson acquired in an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Laboratory (Bar Harbor, ME). Studies were approved by the University of Scientific) using 10 tandem mass spectrometry scans following each sur- Vermont’s Institutional Animal Care and Use Committee (protocol no. 12- vey scan over the entire run. The human and E. coli International Protein 018) in accordance with the recommendations in the Guide for the Care Index forward and reverse concatenated databases were queried with and Use of Laboratory Animals by the National Institutes of Health, and SEQUEST software requiring a 2-Da precursor mass tolerance, tryptic efforts were made to minimize suffering. Sodium pentobarbital was ad- peptide matches, +57.02 Da on cysteine residues, and allowing +15.99 Da ministered via i.p. injection for euthanasia, and cells were processed as for oxidation of methionine residues. Using cross-correlation and delta described (4). Bone marrow–derived dendritic cells (DCs) were generated correlation scores, peptide matches were filtered to a false discovery rate of as previously described (31), and CD4+ T cells were isolated from ,0.01%, and when proteins were required to have at least three peptides splenocytes by negative selection (STEMCELL Technologies, Vancouver, identified, there were no remaining hits matching to the reverse database. BC, Canada) (32). RAW 264.7, J774A.1, and HEK293T cells from For in-solution digestion and subsequent analyses, recombinant proteins American Type Culture Collection (Manassas, VA) were maintained in (6 mg) were concentrated using a Savant SpeedVac concentrator (Thermo DMEM (Life Technologies, Grand Island, NY) supplemented with 10% Fisher Scientific) and then reconstituted in 50 mM ammonium bicarbonate FBS (Life Technologies), 1% L-glutamine (Life Technologies), and 13 buffer containing 4% acetonitrile. Proteins were reduced in 100 mM DTT Primocin (InvivoGen). For experiments in which cell supernatants were for 1 h at 56˚C, alkylated in 200 mM iodoacetamide for 45 min at room examined by ELISA, cells were plated at 2.5 3 105 cells per well in 250 ml temperature, and concentrated using a SpeedVac. Protein digestion was of media in a 48-well plate and allowed to grow overnight. The following performed by reconstitution in digestion solution containing 100 mM day, the cells were treated as indicated within the figure legends for each ammonium bicarbonate, 4% acetonitrile, and 6 ng/ml mass spectrometry– experiment. Cell supernatants were harvested at the end of each experiment, grade trypsin (Promega) via incubation at 37˚C for 18 h. Digestion was spun down at 3300 3 g for 10 min to pellet cellular debris, transferred to terminated with the addition of 10% formic acid, and samples were The Journal of Immunology 2379 processed by ZipTip C18 P10 (EMD Millipore). Liquid chromatography– the highest abundance of protein eluted at a time equivalent to a mass spectrometry-based protein identification was performed on a linear ∼72-kDa standard (Fig. 1K), suggesting that native SAA exists as ion trap LTQ Orbitrap Discovery mass spectrometer coupled to a Surveyor a hexamer, as has been previously reported (37). We also found MS Pump Plus (Thermo Fisher Scientific). Tryptic peptides were loaded onto a 100-mm 3 120-mm capillary column packed with MAGIC C18 (5-mm that only those fractions containing abundant quantities of SAA particle size, 20-nm pore size; Michrom Bioresources) at a flow rate of were capable of eliciting TNF-a production (Fig. 1L). These re- 500 nl/min. Separated peptides were introduced into the linear ion trap via sults suggest that the stimulatory capacity of E. coli–derived SAA ∼ a nanospray ionization source and a laser-pulled 3-mm orifice with a is associated with the native protein and cannot be physically spray voltage of 1.8 kV. Standard “top-ten” data-dependent acquisition was used, in which an Orbitrap survey scan from m/z 360–1600 at 30,000 separated by size-exclusion chromatography. resolution was paralleled by 10 collision-induced dissociation tandem We next transfected HEK cells with empty vectors or plasmids mass spectrometry scans of the most abundant ions in the LTQ. Product ion encoding human SAA1, collected and concentrated the conditioned spectra were searched in a target-decoy fashion using SEQUEST on Pro- media, and measured production of SAA1 of ∼30–40 ng/ml teome Discoverer 1.4 (Thermo Fisher Scientific) against a curated E. coli (Fig. 2A). However, when exposed to J774 cells, these same database with apoSAA, human SAA1 and apoA1, and mouse SAA1 se- quences incorporated. Search parameters were as follows: 1) full trypsin SAA1-rich culture supernatants were not able to stimulate TNF-a enzymatic activity; 2) two missed cleavages; 3) peptides between molec- production, in contrast to that elicited by the addition of 10 ng/ml ular masses of 350–5000 Da; 4) mass tolerance at 20 parts/million for apoSAA to the conditioned media from empty vector–transfected precursor ions and at 0.8 Da for fragment ions; and 5) dynamic modifi- cells (Fig. 2B). In addition to its induction of proinflammatory cations on methionine (+15.99 Da: oxidation) and static modification on cysteine (+57.02 Da: carbamidomethylation). Filters were applied to limit cytokine production, we (4) and others (38) have reported that the false positive rates to ,1% in the data sets. Assigned protein stimulation of DCs with apoSAA elicits the production of cyto- names and aliases were compared with the UniProt KnowledgeBase (33) kines capable of promoting Th17 differentiation. However, ex- Downloaded from and E. coli Protein Database (34) databases, and the EcoTopic posure of DCs to conditioned media from empty vector– or human “LipoProteome” of EcoGene (35) was used to identify verified and SAA1–transfected HEK cells followed by subsequent polyclonal probable E. coli lipoproteins. Data from the mass spectrometry analysis are + included in Supplemental Table I. stimulation of naive CD4 T cells in the DC-derived media was not able to decrease IFN-g production or promote IL-4 and IL- Statistics 17A production, in contrast to apoSAA (Fig. 2C–E). These results

suggest that the proinflammatory and Th17-inducing effects of http://www.jimmunol.org/ Data were analyzed by one-way ANOVA and Dunnett or Tukey multiple SAA may be limited to those proteins made in E. coli. comparisons tests, or by two-way ANOVA and Tukey multiple comparisons test using GraphPad Prism 7.04 for Windows (GraphPad Software, La Jolla, Human SAA1 proteins made in E. coli stimulate TLR2 CA.). A posttest-corrected p value smaller than 0.05 was considered sta- tistically significant. We (4) and others (5–18) have previously reported that the innate immune-stimulating capacity of apoSAA is not due to contami- nating endotoxin (LPS) stimulating TLR4 but is instead mediated Results by protein in the preparation through stimulation of TLR2. In- –derived recombinant SAA proteins induce E. coli terestingly, the TLR2-stimulating capacity of human SAA1 has proinflammatory cytokine production and Th17 polarization by guest on September 24, 2021 been demonstrated almost exclusively through the use of E. coli– To compare the proinflammatory effects of SAA from different derived hSAA1 proteins either generated by the investigators (6) sources, we purchased recombinant SAA proteins from a number of or purchased from a reputable commercial vendor (5–17). A rel- vendors. apoSAA represents the most-commonly used recombinant atively common posttranslational modification of bacterial pro- form of SAA and was able to dose-dependently induce IL-8, IL-6, teins is through lipidation (acylation), which occurs at specific TNF-a, and IL-1b production from human PBMCs (Fig. 1A–D) cysteine amino acids in the context of appropriate neighboring and IL-8 from human neutrophils (Fig. 1E). Because apoSAA is amino acids, a sequence defined as a lipobox (39). Heterodimers human SAA1 made in E. coli but in which aa 60 and 71 are from of TLR2 and TLR6 signal in response to diacylated lipoproteins, SAA2, we also stimulated human PBMCs and neutrophils with whereas heterodimers of TLR2 and TLR1 signal in response to human SAA1 also made from E. coli by the same vendor. Like triacylated lipoproteins. As apoSAA has been previously reported apoSAA, hSAA1 induced robust proinflammatory cytokine pro- to signal through both TLR1 and 2 heterodimers (7), we trans- duction, whereas human SAA1 produced in HEK cells did not fected HEK cells with TLR1, TLR2, or both TLR1 and TLR2, induce proinflammatory cytokine production from human PBMCs then stimulated the cells with commercially sourced recombinant and neutrophils (Fig. 1A–E). Using TNF-a as an indicator of SAA proteins. E. coli–derived human SAA1 proteins were able to proinflammatory cytokine production, similar results were seen in stimulate TLR2- or TLR1/2-transfected cells and induce the primary mouse splenocytes (Fig. 1F) as well as the RAW (Fig. 1G) production of IL-8, whereas mouse SAA1 or human SAA1 made and J774 (Fig. 1H) mouse cell lines. Consequently, in eukaryotic (HEK) cells were not (Fig. 3). Because the human subsequent studies were conducted using J774 cells. apoSAA has SAA1 and apoSAA protein sequences do not contain a lipobox or been reported by us (4, 36) and others (11, 17) to activate even a cysteine that is required for acylation, these results imply the Nlrp3 inflammasome to induce IL-1b secretion. Whereas the that bacterial lipoproteins may be present in the E. coli–derived addition of apoSAA or the synthetic bacterial lipopeptide SAA1 recombinant proteins and mediate the TLR2-dependent Pam3CSK4 to LPS-primed J774 macrophages augmented IL-1b proinflammatory effects ascribed to SAA. secretion, E. coli–derived mouse SAA1 and HEK-derived human SAA1 did not (Fig. 1I). A dose-response study in J774 macro- Lipoproteins associated with human SAA1 elicit phages revealed that the E. coli–derived human SAA1 proteins proinflammatory cytokine production elicited maximal TNF-a production at 1 mg/ml, whereas the To determine whether bacterial proteins were present in apoSAA, mouse SAA1 produced in E. coli (mSAA1) and HEK-derived we separated the preparation by reducing SDS-PAGE, extracted human SAA1 did not induce TNF-a production even at concen- proteins from 12 gel slices, performed trypsin digestion, and an- trations as high as 10 mg/ml (Fig. 1J). apoSAA was subsequently alyzed tryptic peptides by mass spectrometry. We easily visualized separated into 90 fractions using size-exclusion chromatography on the stained gel a predominant 12-kDa band in the apoSAA prep, (fast protein liquid chromatography [FPLC]). The fractions with as was expected based on its molecular mass (Fig. 4A). An 2380 EUKARYOTE-DERIVED SAA IS NOT PROINFLAMMATORY Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Recombinant SAA produced in E. coli, but not produced from eukaryotic cells, stimulates proinflammatory cytokine production. Primary human PBMCs (A–D) and neutrophils (polymorphonuclear cells [PMNs]) (E) were unstimulated (control) or stimulated with 62, 250, or 1000 ng/ml of SAA from different sources for 24 (PBMC) or 3 h (PMN), after which cytokine concentrations were measured by ELISA. Primary C57BL/6J mouse splenocytes (F), mouse RAW macrophage cells (G), and J774 mouse macrophages (H) were unstimulated (control) or stimulated with 1 mg/ml of SAA from different sources. Culture supernatants were collected 24 h later and TNF-a concentrations were measured by ELISA. J774 mouse macrophages were unstimulated (control) or stimulated for 24 h with 100 ng/ml of LPS, 1 mg/ml of apoSAA, LPS plus 100 ng/ml of the lipopeptide Pam3CSK4 (Pam), LPS plus apoSAA, or LPS plus 1 mg/ml of human SAA1 made in HEK cells, and IL-1b concentrations in culture supernatants were measured by ELISA (I). J774 mouse macrophages were unstimulated (control) or stimulated for 24 h with 10, 100, or 1000 ng/ml of SAA from different sources, and TNF-a concentrations in culture supernatants were measured 24 h later by ELISA (J). Two hundred and fifty micrograms of apoSAA was separated by FPLC using a size-exclusion column, and 90 fractions were collected (K). Fractions obtained from FPLC were diluted 1:10 in serum-free media and used to stimulate J774 cells for 24 h, after which TNF-a concentrations were measured by ELISA (L). Data are mean 6 SEM and are representative of three independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett test for multiple comparisons. ****p , 0.0001, **p , 0.01, *p , 0.05. identical pattern was observed by the more sensitive technique of We next sought to determine whether lipoproteins were necessary silver staining (data not shown), indicating that there were no and sufficient for the proinflammatory effects of human SAA1. other proteins present besides SAA that were particularly abun- Therefore, we treated LPS, the triacylated lipopeptide Pam3CSK4, dant. Although the only human protein sequence present in our or apoSAA with LPL, which deactivates the capacity of bacterial analysis was SAA1, the gel lane also contained fragments from 91 proteins to stimulate TLR2 (40). Although LPL had no impact on E. coli proteins, 15 of which were probable or predicted lipo- LPS (Fig. 5A), LPL dose-dependently inhibited the capacity of both proteins (Fig. 4B). Furthermore, mass spectrometry analysis of Pam3CSK4 (Fig. 5B) and apoSAA (Fig. 5C) to stimulate TNF-a tryptic peptides from additional commercial sources of SAA1 production from J774 macrophages. We next exposed OVA or hu- showed that only human SAA1 proteins derived from E. coli man SAA1 produced in HEK cells to Pam3CSK4, subjected the contained bacterial lipoproteins, whereas mouse SAA1 made from preparations to centrifugation through 3-kDa cutoff filters to remove E. coli, human SAA1 made in HEK cells, and apoA1 made from the 1.5-kDa Pam3CSK4, and stimulated J774 cells with the unfil- E. coli by the same manufacturer as the E. coli–derived apoSAA tered preparation or the retained (filtered) fractions that were and hSAA1 did not contain E. coli lipoproteins (Fig. 4C, 4D). readjusted to the original volume. Although neither human SAA1 These data implicate the selectivity of human SAA1 for inter- nor OVA stimulated TNF-a production, and the unfiltered prepa- acting with specific bacterial proteins and lipoproteins, and they rations containing Pam3CSK4 all stimulated TNF-a production, the imply the possibility that this activity is part of the protein’s filtered fractions from Pam3CSK4 incubated with human SAA1, proinflammatory function. but not from Pam3CSK4 incubated with OVA, stimulated TNF-a The Journal of Immunology 2381 Downloaded from http://www.jimmunol.org/

FIGURE 2. apoSAA, but not SAA1 produced from eukaryotic cells, induces TNF-a secretion from macrophages and promotes IL-17A production from CD4 T cells. HEK cells were transiently transfected with empty vector (pcDNA3) or plasmids encoding human SAA1 (hSAA1.pcDNA3 or hSAA1. pCMV6XL5). Forty-eight hours later, culture supernatants were collected and concentrated using Amicon Ultra centrifugal concentrators, SAA1 con- centrations were measured by ELISA (A), and the culture supernatants were used to stimulate J774 cells for 24 h (in the absence or presence of 10 ng/ml by guest on September 24, 2021 apoSAA), after which TNF-a production was measured by ELISA (B). Similarly prepared HEK supernatants were added to bone marrow–derived DCs (BMDCs) for 48 h, their culture supernatants were collected and added to CD4+ T cells that were stimulated with 5 mg/ml immobilized anti-CD3 and 2 mg/ml soluble anti-CD28 for 72 h, after which IL-4 (C), IFN-g (D), and IL-17A (E) were measured by ELISA. Data are mean 6 SEM and are representative of three (A and B)ortwo(C–E) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVAwith Dunnett (A and B)orTukey(C–E) test for multiple comparisons. *p , 0.05, ****p , 0.0001. production (Fig. 5D). These results demonstrate that human SAA1 mediators (5–18), yet several other reports demonstrate no produced from eukaryotic cells can bind lipopeptides that stimulate proinflammatory effect of the endogenous protein enriched from proinflammatory cytokine production. human serum (19, 20) or in transgenic mice producing high levels of circulating human (20, 22) or mouse (21) SAA1. Our own Discussion previous work using recombinant apoSAA demonstrated that Studies conducted using recombinant SAA made in E. coli have the proinflammatory activity of human SAA1 is mediated implicated this family of acute-phase proteins as proinflammatory by TLR2 and is inhibitable by proteinase K, and is present in

FIGURE 3. apoSAA stimulates proinflammatory cytokine production via TLR2. HEK cells were exposed to a transfection reagent (PEI) or were transiently transfected with plasmids encoding hu- man TLR1, TLR2, or both TLR1 and TLR2 (TLR1/2). After 48 h, the cells were then unex- posed (control) or stimulated with 1 mg/ml of SAA from different sources for 24 h, and IL-8 was measured by ELISA. Data are mean 6 SEM and are representative of three independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Tukey test for multiple comparisons. ****p , 0.0001. 2382 EUKARYOTE-DERIVED SAA IS NOT PROINFLAMMATORY

stimulating TLR2 and inducing TNF-a production despite LPL. Al- though LPL has activity on both lipoteichoic acids and lipoproteins (40), Gram-negative E. coli do not contain lipoteichoic acid, implying that the proinflammatory activity of hSAA1 is indeed largely con- ferred by a lipoprotein. In addition to their ability to stimulate TLR2, lipoproteins such as palmitate-conjugated albumin can serve as a strong signal for activation of the intracellular pattern recognition re- ceptor, Nlrp3 (41). Although SAA is not directly palmitoylated, per- haps contaminating bacterial (lipo)proteins are also responsible for the Nlrp3-stimulating capacity of apoSAA (4, 11, 17, 36), especially considering the capacity of Pam3CSK4 to augment IL-1b release. The acylation of bacterial lipoproteins occurs at the cell membrane (39), whereas recombinant proteins accumulate in intracellular inclu- sion bodies prior to acylation (42). Consequently, it is likely that the association of hSAA1 and bacterial lipoproteins occurs during pro- cessing to extract the recombinant protein, not during their biosyn- thesis, implying that similar associations may also occur between circulating SAA and bacterial proteins in vivo during infection. In-

terestingly, the E. coli–derived recombinant apoSAA and hSAA1 Downloaded from preparations used in our studies contained OmpA, a bacterial protein previously reported to activate macrophages (43) and DCs (44) via TLR2 and to interact with SAA (27). The E. coli–derived hSAA1 proteins also contained several bacterial lipoproteins, including peri- plasmic methionine binding lipoprotein (MetQ), peptidoglycan-

associated lipoprotein (Pal), DUF3053 family lipoprotein (YiaF) and http://www.jimmunol.org/ the lipoproteins DcrB and AcrA (Supplemental Table I). However, neither apoSAA nor hSAA1 contained the triacylated Braun Lipo- protein that is abundant in the E. coli outer membrane and stimulates TLR2 (45). Furthermore, none of these bacterial proteins were present in the E. coli–derived recombinant mouse SAA1 used in our studies. Our results support several earlier studies implicating that acute- phase SAA lacks proinflammatory activity (19–21). Interestingly, these studies were conducted using SAA enriched from the serum/ FIGURE 4. apoSAA contains bacterial lipoproteins. Fifty micrograms plasma of subjects with rheumatoid arthritis, a sterile inflamma- by guest on September 24, 2021 of apoSAA was reduced, denatured, and separated by PAGE (A). The tory disease, or from cardiac surgery patients and associated with 12 indicated regions were subjected to in-gel digestion with trypsin. HDL. It will be important to examine whether during bacterial The extracted tryptic peptides were analyzed by liquid chromatography– infection the highly induced levels of SAA, some of which exist as tandem mass spectrometry, and the resulting spectra were searched against soluble proteins independent of HDL but can also form HDL- a database for human and E. coli proteins, including predicted and prob- sized complexes with phospholipids (30), interact with bacterial able E. coli lipoproteins (B). Six micrograms of SAA from different sources were digested in-solution and similarly analyzed by mass spec- lipoproteins and gain proinflammatory activity. trometry. Total E. coli proteins identified (C) as well as predicted and The reasons for the distinct differences between human and probable E. coli lipoproteins identified (D) are indicated. Data are from mouse SAA1 proteins to associate with bacterial (lipo)proteins are two independent experiments. uncertain. Despite their evolutionary relatedness based on genomic organization and regulation, the secreted form of human SAA1 has 75% amino acid identity with mouse SAA1 [by Basic Local TLR4-deficient cells and not inhibitable by polymyxin B (4). Alignment Search Tool (46)], which may account for the afore- Consequently, it is widely appreciated that the proinflammatory mentioned differences. However, there is 80% identity between activities of SAA are not a consequence of endotoxin contami- the N-terminal sequences of human SAA1 (aa 11–58) that have nation. Instead, our studies demonstrate that bacterial proteins, been reported to confer SAA’s TLR2-dependent proinflammatory including lipoproteins, associate with hSAA1 produced in E. coli effects (18) and those in mouse SAA1. Although human SAA1 and mediate the activation of TLR2 and Nlrp3 to induce the derived from HEK cells did not promote the Th17 differentiation production of proinflammatory and Th17-promoting cytokines. induced by E. coli–derived human SAA1, it is very interesting that This association between hSAA1 and bacterial lipoproteins ap- SAA1/2 double knockouts elicit diminished responses to the Th17 pears to be necessary and sufficient for the cytokine-inducing induction elicited by Segmented Filamentous Bacteria infection, effects, because even though recombinant apoA1 contained and that high concentrations of recombinant mouse SAA1 some bacterial proteins, they were not lipoproteins or known were able to increase IL-17A and IL-17F production in a DC- TLR2 agonists and were not able to induce TNF-a production independent manner (47). These results suggest additional activ- from J774 macrophages. The ability of LPL to diminish apoSAA- ities of SAA1 in innate and adaptive immunity that are perhaps induced TNF-a production is dose-dependent, significant, and related to cell survival and immunometabolism (48). In fact, en- substantial, but is incomplete. Although reasons for this are un- dogenous SAA1 isolated from human serum that lacked proin- certain, a possibility includes inadequate access of lipoproteins to flammatory effects did enhance the survival of primary human the enzyme’s active site, in contrast to the effective inactivation of neutrophils by promoting antiapoptotic pathways (19). This ability the lipopeptide Pam3CSK4. Additionally, nonlipopeptides, such as of SAA1 to affect inflammatory cells may be due to differences OmpA, are present in the apoSAA preparation that remain capable of between recombinant and endogenous forms of SAA1, differences The Journal of Immunology 2383 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. Lipopeptides associated with SAA confer its ability to stimulate TNF-a production. One hundred nanograms per milliliter LPS (A), 100 ng/ml Pam3CSK4 (Pam, [B]), and 1 mg/ml apoSAA (C) were incubated with LPL and then used to stimulate J774 cells. Culture supernatants were collected 24 h later, and TNF-a concentrations were measured by ELISA. Human SAA1 from OriGene produced in HEK cells (hSAA1) or OVA (Ova) was unexposed or exposed to Pam3CSK4 (Pam) overnight (control), and some of the preparation was subjected to concentration of proteins .3 kDa using Amicon Ultra centrifugal concentrators (filtered). Control and filtered preparations were used to stimulate J774 cells for 24 h, after which TNF-a concentrations in culture media were measured by ELISA (D). Data are mean 6 SEM and are representative of three (A–C)ortwo(D) independent experiments with n = 4 per group. Statistics were analyzed using one-way ANOVA with Dunnett test for multiple comparisons (A–C) or two-way ANOVA with Tukey test for multiple comparisons (D). ****p , 0.0001 compared with Pam (B), apoSAA (C), or control (D). ###p , 0.001 compared with unfiltered Pam + hSAA1 (D). between the responses of primary cells in the setting of acute with some of the mass spectrometry analyses. We thank Dr. Renee Stapleton inflammation in vivo, differential SAA isoform expression at sites and Sara Ardren for assistance in procuring human blood samples. of inflammation, and the stimulation of cells through receptors besides TLR2, TLR4, and NLRP3 (e.g., FPR2/FPRL1, SB-R1, Disclosures and RAGE) that have been implied to elicit the effects of SAA. The authors have no financial conflicts of interest. Our results call for a re-evaluation of the proinflammatory effects ascribed to SAA through the use of E. coli–derived recombinant proteins. For researchers requiring a recombinant form of human References 1. Uhlar, C. M., and A. S. Whitehead. 1999. Serum amyloid A, the major vertebrate SAA for experimentation, it is reassuring that a commercial acute-phase reactant. Eur. J. Biochem. 265: 501–523. source made in eukaryotic cells and containing no bacterial pro- 2. Ramadori, G., J. D. Sipe, and H. R. Colten. 1985. Expression and regulation of the teins is available. It is highly recommended that such a eukaryotic murine serum amyloid A (SAA) gene in extrahepatic sites. J. Immunol. 135: 3645–3647. 3. Steel, D. M., G. C. Sellar, C. M. Uhlar, S. Simon, F. C. DeBeer, and cell–derived source of SAA be used for all future studies in which A. S. Whitehead. 1993. A constitutively expressed serum amyloid A protein its potential participation in inflammation is evaluated. gene (SAA4) is closely linked to, and shares structural similarities with, an acute-phase serum amyloid A protein gene (SAA2). Genomics 16: 447–454. 4. Ather, J. L., K. Ckless, R. Martin, K. L. Foley, B. T. Suratt, J. E. Boyson, Acknowledgments K. A. Fitzgerald, R. A. Flavell, S. C. Eisenbarth, and M. E. Poynter. 2011. Serum This paper is in honor of Dr. Edward Burgess, a promising doctoral candi- amyloid A activates the NLRP3 inflammasome and promotes Th17 allergic asthma in mice. J. Immunol. 187: 64–73. date whose life was cut short by malignant mesothelioma and who was post- 5. Chami, B., N. Barrie, X. Cai, X. Wang, M. Paul, R. Morton-Chandra, humously conferred the Ph.D. degree. We thank Marion Weir for helping A. Sharland, J. M. Dennis, S. B. Freedman, and P. K. Witting. 2015. Serum 2384 EUKARYOTE-DERIVED SAA IS NOT PROINFLAMMATORY

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Protein Analyzed Gel slice(s) Organism Protein Gene symbol Description E. coli lipoprotein? non-lipoprotein TLR2 agonist? apoSAA (in-gel) B-I Homo sapiens SAA SAA1 Serum amyloid A1 H Escherichia coli AckA ackA Acetate kinase F Escherichia coli AcpD acpD FMN-dependent NADH-azoreductase H, I Escherichia coli AcrA acrA Multidrug efflux pump subunit AcrA Yes I Escherichia coli Ag43 ag43 Antigen 43 F, G Escherichia coli AhpC ahpC Alkyl hydroperoxide reductase C H Escherichia coli AspG2 aspG2 Alkyl hydroperoxide reductase C C, E, F Escherichia coli AtpD atpD ATP synthase subunit beta D-J Escherichia coli AtpF atpF ATP synthase subunit b G Escherichia coli BlaT blaT Beta-lactamase TEM D, E Escherichia coli DcrB dcrB Protein DcrB Yes G-I Escherichia coli DegP degP Periplasmic serine endoprotease DegP H Escherichia coli DegQ degQ Periplasmic pH-dependent serine endoprotease DegQ G-J Escherichia coli DnaK dnaK Chaperone protein DnaK F Escherichia coli DsbA dsbA Thiol:disulfide interchange protein DsbA G Escherichia coli DsbC dsbC Thiol:disulfide interchange protein DsbC D Escherichia coli EcoT ecoT Ecotin C-E, G, H Escherichia coli FkbA fkbA FKBP-type peptidyl-prolyl cis-trans isomerase FkpA D, F-H Escherichia coli FkbB fkbB FKBP-type 22 kDa peptidyl-prolyl cis-trans isomerase G Escherichia coli FrdB frdB Fumarate reductase iron-sulfur subunit C-I Escherichia coli FtnA ftnA Bacterial non-heme ferritin D, E Escherichia coli Ftsh ftsH ATP-dependent zinc metalloprotease FtsH C, E, G, H Escherichia coli GlnH glnH Glutamine-binding periplasmic protein G-I Escherichia coli GltI gltI Glutamate/aspartate import solute-binding protein G Escherichia coli Gph gph Phosphoglycolate phosphatase E-I Escherichia coli GrpE grpE Protein GrpE G, H Escherichia coli HchA hchA Protein/nucleic acid deglycase 1 H, I Escherichia coli HemX hemX Protein HemX G, H Escherichia coli HisJ hisJ Histidine-binding periplasmic protein H Escherichia coli Hmp hmp Histidine-binding periplasmic protein D-I Escherichia coli Hns hns DNA-binding protein H-NS F-H Escherichia coli HtpX htpX Protease HtpX D Escherichia coli IbpB ibpB Small heat shock protein IbpB A-C Escherichia coli IhfA ihfA Integration host factor subunit alpha A-J Escherichia coli IpyR ipyR Inorganic pyrophosphatase D-H Escherichia coli LuxS luxS S-ribosylhomocysteine lyase D, F-H Escherichia coli MetQ metQ D-methionine-binding lipoprotein MetQ Yes E, F Escherichia coli MprA mprA Transcriptional repressor MprA E, G Escherichia coli NifU nifU Iron-sulfur cluster assembly scaffold protein IscU E, F Escherichia coli NuoE nuoE NADH-quinone oxidoreductase subunit E H Escherichia coli O30398 o30398 Malate dehydrogenase D, F-I Escherichia coli OmpA ompA Outer membrane protein A Yes I Escherichia coli OppA oppA Periplasmic oligopeptide-binding protein D-G Escherichia coli Pal pal Peptidoglycan-associated lipoprotein Yes G Escherichia coli PepE pepE Peptidase E H Escherichia coli PotD potD Spermidine/putrescine-binding periplasmic protein H, I Escherichia coli PpiD ppiD Peptidyl-prolyl cis-trans isomerase D H, I Escherichia coli PstS pstS Phosphate-binding protein PstS C, F, G Escherichia coli Q5MD91 q5md91 Adenylate kinase G-J Escherichia coli Q6Q099 q6q099 60 kDa chaperonin G Escherichia coli RbsB rbsB Ribose import binding protein RbsB A-E, H Escherichia coli Rl10 rl10 Ribose import binding protein RbsB B-I Escherichia coli Rl7 rl7 50S ribosomal protein L7/L12 E-H Escherichia coli RlpA rlpA Endolytic peptidoglycan transglycosylase RlpA Yes F-H Escherichia coli RpiA rpiA Ribose-5-phosphate isomerase A F, G Escherichia coli RpoE rpoE ECF RNA polymerase sigma-E factor F, G Escherichia coli Rrf rrf Ribosome-recycling factor D-F Escherichia coli SecG secG Protein-export membrane protein SecG H Escherichia coli SelD selD Selenide, water dikinase D Escherichia coli SlyB slyB Outer membrane lipoprotein SlyB Yes G Escherichia coli SspB sspB Stringent starvation protein B C, G Escherichia coli Syw syw Tryptophan--tRNA ligase G, H Escherichia coli TalB talB Transaldolase B C-E, G, H Escherichia coli TatE tatE Sec-independent protein translocase protein TatE C, D, F-H Escherichia coli ThiO thiO Thioredoxin 1 E, F, H, I Escherichia coli Tig tig Trigger factor G-I Escherichia coli TolB tolB Tol-Pal system protein TolB H Escherichia coli TolC tolC Outer membrane protein TolC B-I Escherichia coli YaeP yaeP UPF0253 protein YaeP C, D, F-H Escherichia coli YajC yajC Sec translocon accessory complex subunit YajC F-H Escherichia coli YbbN ybbN Uncharacterized protein YbbN D, E, G Escherichia coli YbiS ybiS Probable L,D-transpeptidase YbiS D, F-H Escherichia coli YcdO YcdO Iron uptake system component EfeO E, F Escherichia coli YceB yceB Uncharacterized lipoprotein YceB Yes C Escherichia coli YchN ychn Protein YchN D-F, H Escherichia coli YdjA ydjA Putative NAD(P)H nitroreductase YdjA D, E Escherichia coli YeaY yeaY Uncharacterized lipoprotein YeaY Yes E, G Escherichia coli YebE yebE Inner membrane protein YebE D, E Escherichia coli YedD yedD Uncharacterized lipoprotein YedD Yes D-I Escherichia coli YfgM yfgM UPF0070 protein YfgM F, G Escherichia coli YfiO yfiO Outer membrane protein assembly factor BamD Yes G Escherichia coli YggG yggG Metalloprotease LoiP Yes F, G Escherichia coli YgiB ygiB UPF0441 protein YgiB Yes C, D, G Escherichia coli YhcB yhcB Inner membrane protein YhcB H, I Escherichia coli YhjJ yhjJ Protein YhjJ F, G Escherichia coli YiaF yiaF Uncharacterized protein YiaF Yes D, E, G Escherichia coli YibN yibN Uncharacterized protein YibN D Escherichia coli YifL yifL Uncharacterized lipoprotein YifL Yes C Escherichia coli YihD yihD Protein YihD B-D, F-J Escherichia coli YjgF yjgF 2-iminobutanoate/2-iminopropanoate deaminase C Escherichia coli YmgD ymgD Uncharacterized protein YmgD G-I Escherichia coli YraM yram Penicillin-binding protein activator LpoA Yes apoSAA (in solution) Homo sapiens SAA1 SAA1 Serum Amyloid A1 Escherichia coli AcrA acrA Efflux transporter, RND family, MFP subunit Yes Escherichia coli Adk adk Adenylate kinase Escherichia coli AtpF atpf ATP synthase subunit b Escherichia coli DcrB dcrB Putative uncharacterized protein Yes Escherichia coli FklB fklB Peptidyl-prolyl cis-trans isomerase Escherichia coli FkpA fkpA Peptidyl-prolyl cis-trans isomerase Escherichia coli FtnA ftnA Ferritin-1 Escherichia coli GrpE grpE Protein GrpE Escherichia coli Hns hns DNA-binding protein H-NS Escherichia coli MetQ metQ Lipoprotein Yes Escherichia coli MprA mprA Transcriptional regulator, MarR family Escherichia coli Ndk ndk Nucleoside diphosphate kinase Escherichia coli Pal pal Peptidoglycan-associated lipoprotein Yes Escherichia coli Ppa ppa Inorganic pyrophosphatase Escherichia coli PpiD ppiD Peptidyl-prolyl cis-trans isomerase Escherichia coli UspG uspG Universal stress protein UP12 Escherichia coli YbaY ybaY Putative outer membrane lipoprotein Yes Escherichia coli YbbN ybbN Putative thioredoxin domain-containing protein Escherichia coli YedD yedD Putative uncharacterized protein Yes Escherichia coli YfgM yfgM Putative uncharacterized protein Escherichia coli YiaF yiaF Putative uncharacterized protein Yes Escherichia coli YibN yibN Putative uncharacterized protein Escherichia coli YihD yihD Protein YihD Escherichia coli YmgD ymgD Putative uncharacterized protein human SAA1 (in solution) Homo sapiens SAA1 SAA1 Serum Amyloid A1 Escherichia coli AckA ackA Acetate kinase Escherichia coli AcrA acrA Efflux transporter, RND family, MFP subunit Yes Escherichia coli AhpC ahpC Alkyl hydroperoxide reductase subunit C Escherichia coli AtpF atpF ATP synthase subunit b Escherichia coli AtpH atpH ATP synthase subunit delta Escherichia coli Bfr bfr Bacterioferritin Escherichia coli Cld cld Lipopolysaccharide biosynthesis protein Escherichia coli CysK cysK Cysteine synthase Escherichia coli DcrB dcrB Putative uncharacterized protein Yes Escherichia coli DnaK dnaK Chaperone protein DnaK Escherichia coli EcDH1_0473ecdH1_0473 Putative uncharacterized protein Escherichia coli EcDH1 2405ecdH1_2405 Extracellular solute-binding protein family 5 Escherichia coli Eda eda 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase Escherichia coli Efp efp Elongation factor P Escherichia coli ElaB elaB Regulatory protein AmpE Escherichia coli ElpB clpB ATP-dependent chaperone ClpB Escherichia coli Eno eno Enolase Escherichia coli FklB fklB Peptidyl-prolyl cis-trans isomerase Escherichia coli FtnA ftnA Ferritin-1 Escherichia coli GlnH glnH Cationic amino acid ABC transporter, periplasmic binding protein Escherichia coli GlyA glyA Serine hydroxymethyltransferase Escherichia coli GntY gntY Fe/S biogenesis protein NfuA Escherichia coli GroL groL 60 kDa chaperonin Escherichia coli GrpE grpE Protein GrpE Escherichia coli GrxD grxD Glutaredoxin Escherichia coli Gtp gtp Xanthine phosphoribosyltransferase Escherichia coli Hns hns DNA-binding protein H-NS Escherichia coli IbpA ibpA Small heat shock protein IbpA Escherichia coli Lpp lpp LPP repeat-containing protein Yes Escherichia coli ManA manA Mannose-6-phosphate isomerase Escherichia coli MarR marR Transcriptional regulator, MarR family Escherichia coli Mdh mdh Malate dehydrogenase Escherichia coli MetQ metQ Lipoprotein Yes Escherichia coli NadE nadE NH(3)-dependent NAD(+) synthetase Escherichia coli Ndk ndk Nucleoside diphosphate kinase Escherichia coli NusB nusB N utilization substance protein B homolog Escherichia coli OmpA ompA OmpA domain protein transmembrane region-containing protein Yes Escherichia coli Pal pal Peptidoglycan-associated lipoprotein Yes Escherichia coli PfkB pfkB 1-phosphofructokinase Escherichia coli Ppa ppa Inorganic pyrophosphatase Escherichia coli PpiD ppiD Peptidyl-prolyl cis-trans isomerase Escherichia coli ProE proE RNA polymerase sigma factor Escherichia coli PspB pspB Phage shock protein B Escherichia coli PtsH ptsH Phosphocarrier protein Escherichia coli PurE purE N5-carboxyaminoimidazole ribonucleotide mutase Escherichia coli PurN purN Phosphoribosylglycinamide formyltransferase Escherichia coli PyrI pyrI Aspartate carbamoyltransferase regulatory chain Escherichia coli RdgB rdgB dITP/XTP pyrophosphatase Escherichia coli RlpA rlpA Rare lipoprotein A Yes Escherichia coli RpiA rpiA Ribose-5-phosphate isomerase A Escherichia coli RplL rplL 50S ribosomal protein L7/L12 Escherichia coli RpsG rpsG 30S ribosomal protein S7 Escherichia coli SdhB sdhB Succinate dehydrogenase and fumarate reductase iron-sulfur protein Escherichia coli Slp slp Outer membrane lipoprotein, Slp family Yes Escherichia coli SmpA smpA Outer membrane protein assembly factor BamE Escherichia coli TalB talB Transaldolase Escherichia coli TatB tatB Sec-independent protein translocase protein TatB Escherichia coli TatE tatE Sec-independent protein translocase protein Escherichia coli Tig tig Trigger factor Escherichia coli Tpx tpx Probable thiol peroxidase Escherichia coli TufB tufB Elongation factor Tu Escherichia coli Upp upp Uracil phosphoribosyltransferase Escherichia coli UspG uspG Universal stress protein UP12 Escherichia coli YaeP yaeP UPF0253 protein YaeP Escherichia coli YajC yajC Preprotein translocase subunit YajC Escherichia coli YbaY ybaY Putative outer membrane lipoprotein Yes Escherichia coli YbbN ybbN Putative thioredoxin domain-containing protein Escherichia coli YbiS ybiS ErfK/YbiS/YcfS/YnhG family protein Escherichia coli YedD yedD Putative uncharacterized protein Escherichia coli YfbT yfbT HAD-superfamily hydrolase, subfamily IA, variant 3 Escherichia coli YfgM yfgM Putative uncharacterized protein Escherichia coli YfiD yfiD Autonomous glycyl radical cofactor Escherichia coli YiaF yiaF Putative uncharacterized protein Yes Escherichia coli YibN yibN Putative uncharacterized protein Escherichia coli YjdC yjdC Transcriptional regulator, TetR family Escherichia coli YjgF yjgF Endoribonuclease L-PSP Escherichia coli YoaB yoaB Endoribonuclease L-PSP Escherichia coli YraM yraM Penicillin-binding protein activator LpoA Escherichia coli YrbB yrbB Mammalian cell entry related domain protein Escherichia coli YrbB yrbB Putative uncharacterized protein Escherichia coli ZapA zapA Cell division protein ZapA Escherichia coli ZipA zipA Cell division protein ZipA homolog mouse SAA1 (in solution) Mus musculus SAA1 SAA1 Serum Amyloid A1 Escherichia coli CspA cspA Cold shock protein cspA Escherichia coli MalM malM Maltose operon periplasmic Escherichia coli RpsJ rpsJ 30S ribosomal protein S10 Escherichia coli YqiC yqiC Putative uncharacterized protein human SAA1 from HEK (in solution) Homo sapiens SAA1 Saa1 Serum Amyloid A1 human apoA1 (in solution) Homo sapiens APO-A1 APOA1 Apolipoprotein A-I Escherichia coli AcnB acnB Aconitate hydratase 2 Escherichia coli AhpC ahpC Alkyl hydroperoxide reductase subunit C Escherichia coli AhpF ahpF Alkyl hydroperoxide reductase subunit F Escherichia coli Eno eno Enolase Escherichia coli GlyA glyA Serine hydroxymethyltransferase Escherichia coli GlyS glyS Glycine--tRNA ligase beta subunit Escherichia coli MinD minD Site-determining protein Escherichia coli PflB pflB Formate acetyltransferase Escherichia coli PurC purC Phosphoribosylaminoimidazole-succinocarboxamide synthase Escherichia coli PyrG pyrG CTP synthase Escherichia coli Tpx tpx Probable thiol peroxidase Escherichia coli TufB tufB Elongation factor Tu Escherichia coli UspF uspF Stress-induced protein, ATP-binding protein Escherichia coli YmgD ymgD Putative uncharacterized protein ymgD