HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils

Apta, Bonita HR

Davies, Jessica E

Harper, Matthew T

Apollo (Cambridge)

Contents lists available at ScienceDirectJournal of Immunological Methodsjournal homepage: www.elsevier.com/locate/jimTechnical noteCross-reactivity of anti-HMGB1 antibodies for HMGB2Jessica E. Davies, Bonita H.R. Apta, Matthew T. Harper⁎Department of Pharmacology, University of Cambridge, Cambridge, UKA B S T R A C THMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inﬂammation.Despite their relatively high homology, they may have distinct roles, making it essential to be able to diﬀer-entiate between the two. Here we examine the speciﬁcity of ﬁve commercially-available anti-HMGB1 antibodies.By Western blotting of recombinant proteins and HMGB1−/− mouse embryonic ﬁbroblasts, we identiﬁed onlyone HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting speciﬁcantibodies for HMGB1 and HMGB2 allowed identiﬁcation of distinct HMGB1 and HMGB2 subcellular pools inprimary neutrophils.1. IntroductionHMGB1 and HMGB2 are members of the high mobility group box(HMGB) protein family. Typically, these proteins aid DNA replication,repair and transcription. However, HMGB1 shuttles between the nu-cleus and cytoplasm. Cytosolic HMGB1 can be released from the cellthrough active secretion, or passively released when membrane in-tegrity is compromised (Scaﬃdi et al., 2002). Extracellular HMGB1 hasbeen extensively documented to act as an inﬂammatory cytokine(Yamada and Maruyama, 2006). Although extracellular HMGB2 is re-latively understudied, it may also have extracellular actions (Pusterlaet al., 2009).Despite the high degree of sequence homology, and their structuraland biochemical similarities, HMGB1 and HMGB2 functions are notidentical. In knock-out studies, for example, HMGB1−/− but notHMGB2−/− mice die shortly after birth due to hypoglycaemia(Calogero et al., 1999), while HMGB2−/−males have reduced fertility(Ronfani et al., 2001). In humans, an increased serum level of HMGB1has been implicated in several inﬂammatory and autoimmune diseases(Dupire et al., 2012), whereas overexpression of HMGB2 is associatedwith tumour aggression in certain kidney and breast carcinomas (Kwonet al., 2010). In summary, there are now many recent studies identi-fying novel, non-redundant roles for HMGB1 and HMGB2.A range of monoclonal and polyclonal antibodies have been gen-erated for HMGB1 and HMGB2 and are commercially available. Theseantibodies are often marketed as speciﬁc for HMGB1 or 2. In this study,we screened ﬁve commercially-available HMGB1 antibodies for speci-ﬁcity, evaluated through recombinant proteins and knock-out mouseembryonic ﬁbroblasts. We found that most HMGB1 antibodies alsoreadily detected recombinant HMGB2 to some extent under our ex-perimental conditions. Having identiﬁed speciﬁc antibodies for HMGB1and−2, we found that these proteins have unique cellular locations inprimary human neutrophils but not in human endothelial cells, high-lighting the need to be able to distinguish between these isoforms.2. Materials and methods2.1. Tissue cultureHuman umbilical cord vein endothelial cells (HUVEC; PromoCell; c-12203) were cultured in endothelial cell growth medium (PromoCell, c-22010), containing 35 μg/ml gentamycin, 2% v/v fetal calf serum (FCS)and endothelial cell growth supplements (Promocell, c-39215). WT andHMGB1−/−mouse embryonic ﬁbroblasts (MEF) were kindly providedby Professor S. Lippard (Massachusetts Institute of Technology,Cambridge, MA). HMGB2−/− MEFs were purchased fromHMGBiotech (HM-251). MEFs were cultured in DMEM high glucose(Sigma) with 1% v/v penicillin/streptomycin (Sigma) and 5% v/v FCS.2.2. Western blottingLysates were made in RIPA lysis buﬀer and a protein inhibitorcocktail (Sigma) was added to prevent protein degradation. Prior to gelloading, lysate concentrations were established via Bradford assay. MEFlysates, HUVEC, neutrophil lysates (10 μg) or 1 μg recombinant proteins(ProSpec-PRO-581 & HMGBiotech-HM-153) were separated by SDS-PAGE on 12% acrylamide gels under reducing or non-reducing condi-tions, as indicated in the ﬁgure legend. Proteins were transferred to ahttps://doi.org/10.1016/j.jim.2018.02.006Received 29 November 2017; Received in revised form 26 January 2018; Accepted 13 February 2018⁎ Corresponding author at: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.E-mail address: mth29@cam.ac.uk (M.T. Harper).Journal of Immunological Methods 456 (2018) 72–76Available online 15 February 20180022-1759/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).TPVDF membrane (MerckMillipore). Membranes were blocked for 1 h atroom temperature with 5% w/v BSA (Fraction V; Sigma) in TBST (Tris-buﬀered saline containing 0.1% Tween-20), after which membraneswere incubated with a range of HMGB1 antibodies (see Fig. 1 and S.1)or anti-HMGB2 antibodies (Rabbit monoclonal EPR6301; Abcam Cat#ab124670; RRID: AB_10975355) at 4 °C for overnight incubation. Afterwashing with TBST and a 1 h 5% w/v milk block, membranes wereincubated for 2 h at room temperature with anti-rabbit HRP (Cell Sig-naling Technology Cat# 7074) 1:5000, or anti-mouse HRP (Cell Sig-naling Technology Cat# 7076) 1: 10,000, as appropriate. When ap-propriate, anti-beta actin (Clone 8H10D10; Cell Signaling TechnologyCat# 3700, RRID:AB_2242334) or Anti-GAPDH (Clone 14C10; CellSignaling Technology Cat# 5014, RRID:AB_10693448) were used asloading controls. Images were collected through chemiluminescentsubstrate chemistry (Thermoﬁsher Cat# 34577) and documented on X-ray ﬁlm (Amersham Hyperﬁlm ECL, VWR, Cat# 28–9068-35) anddeveloped using an OptiMax X-ray ﬁlm processor (Protec Medi-zintechnik).2.3. Primary cell isolationNeutrophils were obtained from healthy volunteers who providedtheir informed, written consent (local ethics committee approved).Blood was collected in 4% sodium citrate. To isolate neutrophils, 25mlof blood was layered on 15ml Histopaque-1077. Following cen-trifugation (400×g, 30min, no break), the buﬀy coat layer was re-moved to leave the erythrocyte/neutrophil pellet. The pellet was re-suspended 1:1 v/v in Hanks buﬀered saline solution (HBSS, withoutCa2+ and Mg2+; Sigma), then diluted 1:1 in 2% Dextran-500 (made upin HBSS). Erythrocytes were then left to settle for 20min at roomtemperate before the upper (neutrophil layer) was collected, dilutedand pelleted in HBSS. The cell pellet was resuspended in ddH2O for 30 sFig. 1. Antibody epitope identiﬁcation. A) Schematic highlighting HMGB protein domains and conserved cysteine residues. B) Schematic demonstrating the three redox states of HMGBproteins. C) Human and murine HMGB1 and HMGB2 amino acid alignments, shading is co-ordinated to represent degree of similarity, while unshaded regions indicate a residuediﬀerence. Asterisk (*) deﬁnes conserved cysteine residues. Underlined residues locate the reported epitope regions (or central amino acid) for antibodies used within the screen. Note:catalogue number not clone number used as the antibody reference.Table 1Antibody characteristics. CST, Cell Signaling Technology.Catalogue number Clone Company RRID Species Monoclonal/Polyclonal Epitope reported by vendorAb92310 EPR3506 Abcam AB_10975355 Rabbit Monoclonal IPPKGETKKKFKDP6893 D3E5 CST AB_10827882 Rabbit Monoclonal Alanine 1373935 N/A CST AB_2295241 Rabbit Polyclonal Glycine 174ADI-CSA-614-E KS1 Enzo Life Sciences AB_10631414 Mouse Monoclonal Arginine 70-Glutamine 72MAB1690 115603 R&D Systems AB_2117897 Mouse Monoclonal Not availableAb124670 EPR6301 Abcam AB_10975355 Rabbit Monoclonal Asparagine 7J.E. Davies et al. Journal of Immunological Methods 456 (2018) 72–7673to lyse residual erythrocytes, after which the solution was diluted in50ml HBSS and pelleted for immediate resuspension. Neutrophils wereused within 4 h of isolation.2.4. ImmunoﬂuorescenceHUVEC and MEF were grown to conﬂuence on glass coverslips priorto 4% paraformaldehyde (PFA) ﬁxation for 20min at room tempera-ture. After washing with PBS, cells were permeabilised with 0.1%Triton-X100 for 10min. Neutrophils were ﬁxed (1% PFA) in suspensionto avoid activation with the glass coverslip, and permeabilised asabove. Neutrophils were then left to adhere for a minimum of 2 h at 4 °Cto poly-L-lysine-treated glass coverslips. After PBS washing, all sampleswere incubated with anti-HMGB1 (CST 3935, 1:100) or anti-HMGB2(Ab124670, 1:100) primary antibodies and left overnight in 33% v/vFCS and 0.1% v/v sodium azide. Primary antibodies were removed, andﬂuorophore-conjugated anti-rabbit antibodies (Invitrogen Cat# 11-4839-81) 1:200 were applied for 1 h at room temperature. Fluorescentimages were acquired by confocal scanning laser microscopy (Leica).Microscope settings were optimised and kept constant for each celltype.3. Results3.1. Epitope identiﬁcation of HMGB1 antibodiesHuman amino acid sequences of HMGB1 and HMGB2 were sourcedfrom the National Centre for Biotechnology Information (NCBI). Aminoacid sequences were aligned using the free software, Jalview (Clampet al., 2004) to identify regions of homology (Fig. 1C). Using Jalview, itwas calculated that there is 79% sequence similarity between humanHMGB1 and human HMGB2. Both proteins contain two homologouslysine-rich DNA-binding domains, A-Box and B-Box, and a highly ne-gatively-charged C-terminal tail (Fig. 1A). Within the HMGB proteinthere are three conserved cysteine residues (Fig. 1A). These play a keyrole in producing unique redox forms, (Fig.1B), which regulate thebiological eﬀects of release HMGB proteins (Erlandsson Harris et al.,2012).There were three criteria for choosing antibodies for the screen: 1)the antibody should be commercially and readily available from amainstream scientiﬁc retailer; 2) the antibody should be classiﬁed asspeciﬁc (in particular, there should be no apparent cross-reactivity withHMGB2); 3) there should be minimal overlap wherever possible withepitopes between antibodies used in the screen. Epitope regions forHMGB1 were identiﬁed for each HMGB1 antibody within the screen bycontacting the manufacturers (Table 1). Unfortunately, due to protec-tion of intellectual property many of the immunogen sequences wereFig. 2. HMGB1 and HMGB2-speciﬁc antibody selection. A+B) Western blot of human recombinant (r)HMGB1 and rHMGB2. rHMGB1 was probed under reducing (+) and non-reducing(−) conditions. All membranes were exposed for the same time to ensure comparability. Asterisks (*) on the left-hand side indicate the expected weight of his-tagged rHMGB1 and non-tagged rHMGB2. C) Western blot of wildtype (WT), HMGB1−/− (MEF 1−/−) and HMGB2−/− (MEF2−/−) MEF lysates, under reducing conditions. Images are representative of 3independent repeats.J.E. Davies et al. Journal of Immunological Methods 456 (2018) 72–7674not divulged from the manufacturers. In these cases, centralised aminoacids of the epitopes were given. In the case of the antibody MAB1690,R&D Systems stated that they do not conduct epitope mapping so wereunable to locate the exact address of their antibody.3.2. HMGB1-speciﬁc antibody selectionHis-tagged, recombinant human HMGB1 (~28 kDa) and (untagged)recombinant human HMGB2 (~25 kDa) were subjected to SDS-PAGEand Western blotting with each antibody. All antibodies were used attheir recommended concentration (Fig. S.1). Under our experimentalconditions, MAB1690 and 6893 readily detected HMGB2 in addition toHMGB1 (Fig. 2A). Ab92310 weakly detected HMGB2. More intensebands were observed under reducing conditions against the re-combinant HMGB1, suggesting that all antibodies in this screen dis-played higher aﬃnity for the reduced redox form of HMGB1. The non-reduced protein ran slightly faster, suggesting that the single disulphidebond inﬂuences the globular structure of the protein. Notably, Ab92310could readily detect reduced HMGB1 but was particularly poor at de-tecting non-reduced HMGB1. This means that Ab92310 might not de-tect native HMGB1 if present in its non-reduced form. Some HMGB1antibodies (MAB1690 and 6893) detected prominent bands at around55 kDa and 50 kDa. This may represent dimers or aggregates forming athigh concentrations of recombinant HMGB1 protein. These bands werealso weakly detected by ADI-CSA-614-E, which also detected similarbands with recombinant HMGB2 (despite not detecting monomerHMGB2). Low molecular weight fragments were observed with allHMGB1 antibodies except for 3935. This may represent recombinantprotein degradation products. In most cases these were only observed inreduced samples. Based on these observations, we decided to use 3935as our HMGB1-speciﬁc antibody.Additionally, one HMGB2 antibody was trialled (Ab124670).Thisantibody did not detect HMGB1 under our experimental conditions,either in reduced or non-reduced forms, but readily detected HMGB2,suggesting that it does speciﬁcally detect HMGB2. Interestingly,Ab124670 is reported to bind to an epitope that varies by only oneamino acid between HMGB1 and HMGB2 in both mice and humans(Fig. 1).To test whether these antibodies retained speciﬁcity when used oncell lysates, we took advantage of previously generated knock out MEFHMGB1−/− and HMGB2−/− cell lines (Fig. 2B). Given the highdegree of sequence likeness for human and murine HMGB1 andHMGB2, 99% and 97% respectively (Fig. S.2), it was assumed that MEFcells would be a reliable cell line to work with. Anti-HMGB1 3935readily detected HMGB1 in wild-type (WT) MEFs. This band was absentin HMGB1−/− MEFs, and no other bands were observed. Conversely,anti-HMGB2 Ab124670 readily detected a ~25 kDa protein in WT MEFsthat was absent in HMGB2−/− MEFs. In addition, there was a non-speciﬁc band at ~36 kDa when MEFs were probed with Ab124670.3.3. HMGB1 is diﬀerentially located in neutrophils compared to HMGB2To demonstrate the importance of speciﬁc antibodies for HMGB1and HMGB2, endothelial cells (HUVEC) and primary human neu-trophils were stained to identify HMGB1 and HMGB2 cellular locationsby confocal microscopy. HMGB1 and HMGB2 were both nuclear inHUVEC (Fig. 3A). In contrast, HMGB1 and HMGB2 had distinct loca-tions in neutrophils. HMGB1 in resting neutrophils was predominantlycytosolic and distributed in puncta, whereas HMGB2 was retained inthe nucleus (Fig. 3B). Appropriate IgG controls were carried out (Fig.Fig. 3. Subcellular locations of HMGB1 and HMGB2 are cell type speciﬁc. Confocal microscopy images of HUVEC (A) and neutrophils (B) using a 63× oil immersion lens. HMGB1 (3935)and HMGB2 (EPR6301) proteins are shown in red, and blue is the nuclear stain (DAPI). Scale bar is 50 μm in both. The cell marked with the white arrow in the merge is shown in highermagniﬁcation in the ﬁnal panel. Images are representative of 3 independent repeats. Western blot of HUVEC (C) and neutrophil (D) reduced lysates. 1–3 indicate three diﬀerent lysatesprepared independently. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)J.E. Davies et al. Journal of Immunological Methods 456 (2018) 72–7675S.3). Finally, we tested whether the two antibodies used could speciﬁ-cally detected HMGB1 and HMGB2 in HUVEC and neutrophils byWestern blot (Fig. 3C–D). A single band (~25 kDa) was observed foreach protein in each cell type. Notably, the non-speciﬁc band(~36 kDa) found when MEF lysates were probed with anti-HMGB2(Ab124670) was not seen in either HUVEC or neutrophil lysates. Cy-toplasmic HMGB1 in neutrophils has been previously shown to be dueto mono-methylation of Lys42 (Ito et al., 2007). However, to the best ofour knowledge this is the ﬁrst example of HMGB1 and HMGB2 beingdiﬀerentially localised within the same cell, which leads to speculationthat these 79% homologous proteins may have diﬀerent biologicalfunctions in neutrophils.In conclusion, some commercially-available HMGB1 antibodiescross-reacted with the highly homologous recombinant protein,HMGB2, under our experimental conditions. We selected two speciﬁcHMGB1 and HMGB2 antibodies based on Western blot analysis fromrecombinant proteins and MEF lysates deﬁcient in HMGB1 or HMGB2.By using these speciﬁc antibodies, we demonstrated that althoughHMGB1 and HMGB2 are identically localised to the nucleus in en-dothelial cells, in neutrophils HMGB2 remained nuclear and HMGB1cytoplasmic. This suggests that HMGB1 and 2 may have distinct roles inneutrophils, and highlights the importance of antibody speciﬁcity,especially when working with primary cells which are not geneticallytractable.AcknowledgementsWe acknowledge Professor S. Lippard at the Massachusetts Instituteof Technology, Cambridge, Massachusetts for supplying the WT andHMGB1−/− MEFS. This research was funded by the British HeartFoundation, grant number PG/16/45/32152.DisclosureAll authors declare that there is no conﬂict of interest regarding thepublication of this paper.AuthorshipJ. E. Davies, conceived and designed experiments, acquired dataand drafted the manuscript. B.H.R. Apta, acquired data and revised themanuscript. M. T. Harper, conceived the work and revised the manu-script.Appendix A. Supplementary dataSupplementary data to this article can be found online at https://doi.org/10.1016/j.jim.2018.02.006.ReferencesCalogero, S., Grassi, F., Aguzzi, A., Voigtländer, T., Ferrier, P., Ferrari, S., Bianchi, M.E.,1999. The lack of chromosomal protein Hmg1 does not disrupt cell growth but causeslethal hypoglycaemia in newborn mice. Nat. Genet. 22, 276–280.Clamp, M., Cuﬀ, J., Searle, S.M., Barton, G.J., 2004. The Jalview Java alignment editor.Bioinforma. Appl. NOTE 20, 426–427. http://dx.doi.org/10.1093/bioinformatics/btg430.Dupire, G., Nicaise, C., Gangji, V., Soyfoo, M., 2012. Increased serum levels of high-mobility group box 1 (HMGB1) in primary Sjögren's syndrome. Scand. J. Rheumatol.41, 120–123. http://dx.doi.org/10.3109/03009742.2011.633099.Erlandsson Harris, H., Andersson, U., Pisetsky, D.S., 2012. HMGB1: a multifunctionalalarmin driving autoimmune and inﬂammatory disease. Nat. Publ. Group 8. http://dx.doi.org/10.1038/nrrheum.2011.222.Ito, I., Fukazawa, J., Yoshida, M., 2007. Post-translational methylation of high mobilitygroup box 1 (HMGB1) causes its cytoplasmic localization in neutrophils. J. Biol.Chem. 282, 16336–16344. http://dx.doi.org/10.1074/jbc.M608467200.Kwon, J.-H., Kim, J., Park, J.Y., Hong, S.M., Park, C.W., Hong, S.J., Park, S.Y., Choi, Y.J.,Do, I.-G., Joh, J.-W., Kim, D.S., Choi, K.Y., 2010. Overexpression of high-mobilitygroup box 2 is associated with tumor aggressiveness and prognosis of hepatocellularcarcinoma. Clin. Cancer Res. 16, 5511–5521. http://dx.doi.org/10.1158/1078-0432.CCR-10-0825.Pusterla, T., de Marchis, F., Palumbo, R., Bianchi, M.E., 2009. High mobility group B2 issecreted by myeloid cells and has mitogenic and chemoattractant activities similar tohigh mobility group B1. Autoimmunity 42, 308–310. http://dx.doi.org/10.1080/08916930902831845.Ronfani, L., Ferraguti, M., Croci, L., Ovitt, C.E., Scholer, H.R., Consalez, G.G., Bianchi,M.E., 2001. Reduced fertility and spermatogenesis defects in mice lacking chromo-somal protein Hmgb2. Development 128, 1265–1273.Scaﬃdi, P., Misteli, T., Bianchi, M.E., 2002. Release of chromatin protein HMGB1 bynecrotic cells triggers inﬂammation. Nature 418, 191–195. http://dx.doi.org/10.1038/nature00858.Yamada, S., Maruyama, I., 2006. HMGB1, a novel inﬂammatory cytokine. Clin. Chim.Acta 36–47. http://dx.doi.org/10.1016/j.cca.2006.07.019.J.E. Davies et al. Journal of Immunological Methods 456 (2018) 72–7676

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

https://www.repository.cam.ac.uk/bitstream/handle/1810/275439/1-s2.0-S0022175917305070-main.pdf?sequence=5&isAllowed=y

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

Abstract

Similar works

Full text

Available Versions

Apollo (Cambridge)