Identification of SERPINB1 As a Physiological Inhibitor of Human Granzyme H

This information is current as Li Wang, Qian Li, Lianfeng Wu, Shengwu Liu, Yong of October 1, 2021. Zhang, Xuan Yang, Pingping Zhu, Honglian Zhang, Kai Zhang, Jizhong Lou, Pingsheng Liu, Liang Tong, Fei Sun and Zusen Fan J Immunol 2013; 190:1319-1330; Prepublished online 26 December 2012; doi: 10.4049/jimmunol.1202542 Downloaded from http://www.jimmunol.org/content/190/3/1319

Supplementary http://www.jimmunol.org/content/suppl/2013/01/04/jimmunol.120254 http://www.jimmunol.org/ Material 2.DC1 References This article cites 35 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/190/3/1319.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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of SERPINB1 As a Physiological Inhibitor of Human Granzyme H

Li Wang,*,1 Qian Li,*,1 Lianfeng Wu,* Shengwu Liu,* Yong Zhang,† Xuan Yang,* Pingping Zhu,* Honglian Zhang,* Kai Zhang,† Jizhong Lou,† Pingsheng Liu,† Liang Tong,‡ Fei Sun,† and Zusen Fan*

The granzyme/perforin pathway is a major mechanism for cytotoxic lymphocytes to eliminate virus-infected and tumor cells. The balance between activation and inhibition of the proteolytic cascade must be tightly controlled to avoid self damage. Granzyme H (GzmH) is constitutively expressed in NK cells and induces target cell death; however, how GzmH activity is regulated remains elusive. We reported earlier the crystal structures of inactive D102N-GzmH alone and in complex with its synthetic substrate and inhibitor, as well as defined the mechanisms of substrate recognition and enzymatic activation. In this study, we identified SERPINB1 343

as a potent intracellular inhibitor for GzmH. Upon cleavage of the reactive center loop at Phe , SERPINB1 forms an SDS-stable Downloaded from covalent complex with GzmH. SERPINB1 overexpression suppresses GzmH- or LAK cell–mediated cytotoxicity. We determined the crystal structures of active GzmH and SERPINB1 (LM-DD mutant) in the native conformation to 3.0- and 2.9-A˚ resolution, respectively. Molecular modeling reveals the possible conformational changes in GzmH for the suicide inhibition. Our findings provide new insights into the inhibitory mechanism of SERPINB1 against human GzmH. The Journal of Immunology, 2013, 190: 1319–1330.

atural killer cells and CTLs play important roles in im- conserved structural architectures but different substrate specific- http://www.jimmunol.org/ mune surveillance against virus-infected or transformed ities. Five human Gzms (A, B, H, K, and M) have been identified N tumor cells. Granzyme (Gzm)/perforin-mediated cell in cytotoxic granules, which induce cell death to eliminate virus- death is a major pathway for killer cells to destroy their target cells infected or tumor cells by proteolysis of intracellular target sub- (1, 2). Gzm are members of the serine protease family with highly strates (1). Their response activities must be tightly controlled. Lack of Gzm activity lowers immune responsiveness, leading to infection and cancer. Overactivity of Gzm results in cellular damage *Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of † and tissue destruction. However, how Gzm activity is modulated Biophysics, Chinese Academy of Sciences, Beijing 100101, China; National Labora- remains elusive. tory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, by guest on October 1, 2021 Beijing 100101, China; and ‡Department of Biological Sciences, Columbia Univer- Gzm H (GzmH)is constitutively expressedinNK cells,suggesting sity, New York, NY 10027 that it plays a critical role in NK cell–mediated immunity (3–5). We 1L. Wang and Q.L. contributed equally to this work. (6) and Fellows et al. (3) demonstrated that GzmH induces caspase- Received for publication September 10, 2012. Accepted for publication November dependent and -independent cell death with DNA degradation. 28, 2012. GzmH can directly proteolyze two adenoviral essential for This work was supported by the National Natural Science Foundation of China (30830030, viral replication: the adenovirus DNA-binding and the ad- 31128003, 31170837, 31021062, and 30972676), the 973 Program (2010CB911902), the Innovative program of the Chinese Academy of Sciences (XDA01010407), and enovirus 100K assembly protein for virus assembly (7). GzmH is the KC Wong Education Foundation (Hong Kong). also able to cleave the multifunctional phosphoprotein La to abro- L. Wang and Q.L. designed and performed research, analyzed data, and wrote the gate hepatitis C virus–internal ribosome entry site–mediated trans- manuscript; L. Wu and P.L. contributed to the granule isolation; S.L., P.Z., and H.Z. lational activity (8). We recently showed that GzmH participates in performed cytolysis assays; Y.Z. and J.L. performed structure modeling; X.Y. and K.Z. contributed to protein purification and crystallization; L.T. and F.S. supervised the clearance of hepatitis B virus (HBV) via cleavage of the HBx all crystallographic studies and analyses; and Z.F. initiated the research, analyzed protein (9). Adoptive transfer of GzmH-expressing NK cells into an data, and wrote the manuscript. HBV mouse model augments HBV clearance. Notably, low GzmH Address correspondence and reprint requests to Drs. Liang Tong, Fei Sun, or Zusen expression in cytotoxic lymphocytes is correlated with susceptibility Fan, Department of Biological Sciences, Columbia University, New York, NY 10027 (L.T.); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese to HBV infection in individuals. We also recently reported the crystal Academy of Sciences, Beijing 100101, China (F.S.); or Chinese Academy of Sciences structure of the enzymatically inactive D102N-GzmH mutant and Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy defined the substrate specificity of GzmH (10). of Sciences, 15 Datun Road, Beijing 100101, China (Z.F.). E-mail addresses: ltong@ columbia.edu (L.T.), [email protected] (F.S.), and [email protected] (Z.F.) Serine protease inhibitors () are a class of proteins that is The atomic coordinates and structure factors have been deposited in the Protein Data primarily known to irreversibly suppress the proteolytic activities Bank (http://www.rcsb.org) under accession numbers 4GA7 for LM-DD-SERPINB1 of serine proteases. The native inhibitory fold contains a and 4GAW for wtGzmH. variable reactive center loop (RCL), which is the primary site of The online version of this article contains supplemental material. association with target proteases (11). Upon cleavage at the P1 site Abbreviations used in this article: CMA, concanamycin A; Gzm, granzyme; GzmB, in the RCL, serpins undergo a conformational change by which granzyme B; GzmH, granzyme H; GzmM, granzyme M; HBV, hepatitis B virus; ICAD, inhibitor of caspase-activated DNase; pAb, polyclonal Ab; PEG3350, poly- the serpin and the target protease form a covalent complex to ethylene glycol 3350; RCL, reactive center loop; RT, room temperature; serpin, block the enzymatic activity. SERPINB9 was identified as an in- serine protease inhibitor; SI, stoichiometry of inhibition; siRNA, small interfering tracellular inhibitor of Gzm B (GzmB) (12). SERPINB4 is an in- RNA; wt, wild-type. hibitor of Gzm M (GzmM) and suppresses GzmM-mediated cell Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 death (13). However, no physiological intracellular inhibitors have www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202542 1320 SERPINB1 INHIBITS THE ACTIVITY OF HUMAN GRANZYME H been defined for human GzmH. In this study, we identified that His tag. SERPINB1 RCL-mutated plasmids were generated using the SERPINB1 is an intracellular inhibitor of human GzmH. GzmH QuickChange site-directed mutagenesis kit (Stratagene). All constructs cleaves SERPINB1 at Phe343 in the RCL to mediate suicide inhi- were confirmed by DNA sequencing. His-tagged wild-type wtSERPINB1, F343A-SERPINB1,andLM-DD-SERPINB1 were generated and purified bition. We also identified the crystal structures of the full-length using Ni-NTA resin for 63His-tagged proteins, Resource Q Sepharose anion SERPINB1 molecule and the active form of human GzmH, pro- exchange, and, finally, Superdex 75 gel-filtration chromatography (GE viding structural insights into the inhibitory mechanism. Healthcare). wtGzmH and D102N-GzmH proteins were overexpressed in Escherichia coli Rosetta (DE3) as inclusion bodies and then refolded and purified, as previously described (10). Materials and Methods Cell lines, Abs, and reagents Pull-down assay m 2+ The human NK tumor cell line YTS, the human T cell lymphoblast-like cell For Ni-NTA pull-down assay, 50 l His Resin was charged by Ni , bal- anced, and incubated with 200 ng wtSERPINB1 in binding buffer con- line Jurkat, and the MHC class I–negative mutant B lymphoblastoid cell line taining 40 mM Tris (pH 8), 200 mM NaCl, 10 mM imidazole, and 10% 721.221 (provided by Dr. Baoxue Ge, Institute of Health Science, Chinese Academy of Sciences) were maintained in RPMI 1640 medium supple- glycerol. SERPINB1-attached Ni-NTA His beads were incubated with mented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. purified D102N-GzmH at 4˚C for 2 h. For Affi-Gel10 conjugated pull- Commercial Abs used included mouse mAb against GzmH, mouse mAb down assay, 200 ng D102N-GzmH was conjugated with Affi-Gel10 beads against perforin, rabbit polyclonal Ab (pAb) against EEA1, rabbit pAb and then incubated with purified wtSERPINB1 in binding buffer containing against LAMP1 (Abcam), rabbit pAb against La (Cell Signaling Tech- 40 mM HEPES (pH 7.5), 200 mM NaCl, and 10% glycerol at RT for 30 min. After washing with a binding buffer, the beads were resolved in loading nology), goat pAb against SERPINB1, rabbit antiserum against NM23-H1 buffer and boiled for 10 min, and bound proteins were detected by Western (Santa Cruz Biotechnology), b-actin (Sigma-Aldrich), Alexa Fluor 488– conjugated goat anti-rat IgG (BioLegend), Alexa Fluor 594–conjugated blotting. Downloaded from donkey anti-mouse IgG, Alexa Fluor 488–conjugated donkey anti-mouse Gel-filtration chromatography IgG, Alexa Fluor 594–conjugated donkey anti-rabbit IgG (Molecular Probes), and HRP-conjugated secondary Abs (Santa Cruz Biotechnology). Gel-filtration chromatography was performed on a Superdex 75 column by Rabbit antiserum against inhibitor of caspase-activated DNase (ICAD) was HPLC (GE Healthcare). The column was equilibrated with two column kindly provided by Dr. R.P. Sekaly (Laboratoire d’Immunologie, Uni- volumes of buffer A (40 mM Tris [pH 8], 150 mM NaCl). A mixture of versite´ de Montreal, Montreal, QC, Canada). SERPINB1 anti-mouse and purified wtSERPINB1 and D102N-GzmH proteins was incubated at a molar SERPINB1 anti-rat polyclonal antisera for confocal microscopy were ratio of 1:2 at 4˚C for 5 h, or the individual protein was separately injected generated by our laboratory and were specific for recognition of human into the column running with buffer A. Samples were fractionated with a http://www.jimmunol.org/ SERPINB1 determined by immunoblotting (data not shown). flow rate of 0.5 ml/min and collected as 0.5-ml fractions after injection into the column. Protein fractions were separated by SDS-PAGE on 12% gels. Granule isolation from YTS cells Detection of complex formation A total of 1 3 109 YTS cells was washed three times in ice-cold PBS and resuspended into 20 ml relaxation buffer (100 mM KCl, 1.25 mM EGTA, 10 wtSERPINB1 and GzmH proteins were incubated at 37˚C in 25 mM Tris mM PIPES, 3.5 mM MgCl2). ATP (1 mM) was added just before use. The (pH 8), 150 mM NaCl for the indicated time points, with or without cells were disrupted on ice using a nitrogen bomb at 450 c with N2 for 25 synthesized GzmH inhibitor (10). YTS cell lysates were incubated at 37˚C min (Parr Instrument Company Model 4639 Cell Disruption Vessel). The for the indicated times. The reactions were stopped by 53 SDS loading homogenates were separated from pellet nuclei, mitochondria, and the buffer. The samples were then boiled for 10 min, resolved by SDS-PAGE, remaining intact cells by centrifugation at 400 3 g at 4˚C for 10 min. The and detected by Coomassie Brilliant Blue staining or immunoblotting by guest on October 1, 2021 final supernatant was postnuclear supernatant, which was centrifuged at with the indicated Abs. 15,000 3 g for 10 min to collect the enriched organelles. At the same time, 80 ml 40% (w/v) Percoll was added equally to four SW70 tubes and Knockdown of GzmH and SERPINB1 in YTS cells centrifuged at 20,000 rpm for 10 min to form Percoll gradients. The enriched organelles were resuspended in 20 ml relaxation buffer and layered carefully RNA sequences against GzmH (59-GATCCCCGTGTGAACGTCTCTTC- above the preformed Percoll gradient. The tubes were centrifuged for 35 min CATTTCAAGAGAATGGAAGAGACGTTCACACTTTTTA-39 [sense] and at 20,000 rpm. Twenty-four 1-ml fractions were collected from top to bottom 59-GCTTAAAAAGTGTGAACGTCTCTTCCATTCTCTTGAAATGGA- and analyzed by SDS-PAGE and Western blotting. The granule-containing AGAGACGTTCACACGGG-39 [antisense]) were designed based on pSUPER fractions were centrifuged overnight at 22,000 rpm (SW40 tube) to pellet system instructions (Oligoengine) and cloned as previously reported (14). Percoll. Granules formed a thin layer above the Percoll pellet; they were For production of lentivirus, 293T cells were seeded onto a 10-cm dish and collected and washed three times with relaxation buffer. The granules were transfected with 4 mg transfer vector pSIN-EF2-shGzmH, 4 mg gag-pol, and resuspended in 50 mM Tris buffer and disrupted by at least three freeze–thaw 2 mg envelop plasmids using Lipofectamine 2000 (Invitrogen). After 48 h cycles in liquid nitrogen and then centrifuged at 14,800 rpm for 20 min to of incubation, culture supernatants were harvested, passed through a 0.45-mm separate the soluble proteins and membrane proteins. filter, and concentrated by ultracentrifugation at 20,000 rpm for 2 h. YTS cells were infected by lentivirus in the presence of 8 mg/ml Polybrene. Stable Confocal microscopy clone pools were selected and maintained in the presence of 1 mg/ml pu- romycin. For SERPINB1 silencing, SERPINB1 and scrambled control small Cultured YTS cells (105) were washed three times with PBS and spread interfering RNA (siRNA) consisting of 21 bp with a two-base deoxy- onto poly-L-lysine–coated glass slides (Sigma-Aldrich) for 5 min. The nucleotide overhang were purchased from GenePharma (Shanghai, China). slides were rinsed twice to remove nonadherent cells. Cells adhering to the The following sequences were used: siSERPINB1, sense: 59-GGCGUUG- slide were fixed with 4% paraformaldehyde for 30 min and permeabilized AGUGAGAACAAUTT-39; antisense: 59-AUUGUUCUCACUCAACGCC- with 0.5% Nonidet P-40 in PBS for 5 to 10 min at room temperature (RT). TT-39 and control, sense: 59-UUCUCCGAACGUGUCACGUTT-39; anti- After 30 min of blocking with 10% donkey serum (Life Technologies), sense: 59-ACGUGACACGUUCGGAGAATT-39. Transient transfection of cells were stained with the primary Abs at 4˚C overnight. Then the samples YTS cells was performed with Cell Line Nucleofector Kit R (Amaxa). A were rinsed three times with PBS and incubated with appropriate sec- total of 2 3 106 YTS cells was pelleted at 100 3 g, resuspended in 100 ml ondary Abs. Images were acquired with an Olympus LSCMFV500 laser transfection solution, and nucleofected with Amaxa program O-017. The scanning confocal microscope. samples were added to prewarmed recovery medium and incubated in a six- well dish. The knockdown efficiency was detected by immunoblotting. Protein expression and purification Cleavage assay and detection of stoichiometry of inhibition The human SERPINB1 cDNA was amplified from the YTS cDNA library using specific primers (forward: 59-CGTGCCTCGGCGGCCTGTC-39; 721.221 cell lysates (1 3 105) were incubated with wtGzmH (0.4 mM) and reverse: 59-GGCTCTATTAAAAACTCAGGT-39). A nested PCR was per- the indicated amounts of wtSERPINB1 at 37˚C for 2 h in 20 ml cleavage formed using the cDNA templates from first-round PCR with primers buffer. The reactions were terminated and subjected to immunoblotting, as (forward: 59-AGAATTCATGGAGCAGCTGAGCTCAG-39;reverse:59-TA- described (10). For stoichiometric determinations, 100 nM Gzm was pre- CTCGAGTAAGGGGAAGAAAATCTC-39). The full-length SERPINB1 incubated separately with different concentrations of wtSERPINB1, as indi- was cloned into the pET-15b vector (Novagen) with an N-terminal cated by GzmH to wtSERPINB1 molor ratios in a range from 0 to 2, at 37˚C The Journal of Immunology 1321 for 1 h in 25 mM Tris (pH 8), 150 mM NaCl, and relevant synthetic substrates tivity was measured as described previously (9). For the inhibition assay, (300 mM) were added to the samples and diluted to 100 ml for an additional LAK cells were pretreated with 500 nM concanamycin A (CMA) for 2 h or 30-min incubation at 37˚C. The remaining enzyme activity was determined with 300 mM GzmH inhibitor for 1 h before incubation with target cells. by measuring absorbance at 405 nm using a Multilabel Counter (Wallac 1420 Victor; PerkinElmer). The corresponding substrates were as follows: Ac- Crystallization and data collection PTSY-pNA for GzmH, Suc-VANR-pNA for Gzm A, Z-IEPD-AFC for GzmB, Ac-YRFK-pNA for Gzm K, and Suc-AAPL-pNA for GzmM. Initial hits were obtained using commercially available sparse matrix screens (Hampton Research) in the hanging drop vapor-diffusion method at 16˚C. GzmH loading and LAK cell–mediated cytolysis assay Optimization was conducted and diffraction-quality crystals of LM-DD- wtSERPINB1 and F343A-SERPINB1 were cloned into the pSIN-EF2 vector SERPINB1 were eventually obtained in 0.18 M lithium citrate tribasic and then lentiviruses were produced as described above. Jurkat cells were tetrahydrate, 18% (w/v) polyethylene glycol 3350 (PEG3350), with a pro- infected with the described lentivirus, and stable clone pools were selected tein concentration near 8 mg/ml. Crystals were cryoprotected in reservoir and maintained in the presence of 1 mg/ml puromycin. Jurkat cells stably solution supplemented with 30% (w/v) PEG3350 and then flash frozen in liquid nitrogen. The data set was obtained on the Shanghai Synchrotron transfected with control vector, wtSERPINB1, and F343A-SERPINB1 were ˚ washed twice in serum-free RPMI 1640, and then 1 mMGzmHor1mM Radiation Facility beamline BL17U (l = 0.98 A) at 100 K. Crystals of GzmB was loaded into Jurkat cells with adenovirus at 37˚C for 4 h. Then wtGzmH were obtained in a solution containing 0.2 M Li2SO4,0.1M Bicine (pH 8.5), 25% (w/v) PEG3350. Data sets were collected at 100 K at cells were harvested and stained with FITC-conjugated annexin V and ˚ propidium iodide (Bender Med Systems), followed by flow cytometry the beamline BL5A (l = 0.9A) of the Photon Factory (Tsukuba, Japan). All (FACSCalibur; BD Biosciences). Data were analyzed using FlowJo software diffraction data were processed using the HKL-2000 suite of programs (15). (TreeStar). PBMCs from healthy donors were purchased from the Beijing Structure determination and refinement Blood Center and isolated by Ficoll-Hypaque gradient centrifugation. For generation of LAK cells, PBMCs were cultured in RPMI 1640 medium The structure of SERPINB1 was solved by molecular replacement with supplemented with 15% FBS (Life Technologies) with 500 U/ml recombi-

PHASER (16), using plasminogen activator inhibitor-2 as the search model Downloaded from nant human IL-2 for 4 d. Jurkat cells stably transfected with control vector, (PDB code 1BY7). The structure of wtGzmH was solved by molecular wtSERPINB1, and F343A-SERPINB1 were labeled with 200 mCi 51Cr for replacement with COMO using the structure of D102N-GzmH as a search 1 h. LAK cells were added to 51Cr-labeled Jurkat cells at an E:T ratio of 1. model (PDB code 3TK9). Structure refinement was carried out using CNS After 4 h of incubation, the supernatants were harvested, and the radioac- and REFMAC, and COOT was used for manual model building (17). Final http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. SERPINB1 is fractionated with GzmH from the separation of cytotoxic granules. (A) Diagram of isolation steps of secretory lysosomes from the NK cell line YTS. YTS cells were disrupted by nitrogen decompression, and the secretory lysosomes were separated from other organelles by Percoll- gradient fractionation. The product of each step is abbreviated in parentheses. (B) Soluble components in the isolated secretory lysosomes were identified by mass spectrometry. Secretory lysosomes were disrupted by three freeze–thaw cycles. Supernatants (S2) were subjected to SDS-PAGE and visualized by Coomassie Brilliant Blue staining (upper panel). Arrows denote three major bands. Peptide sequencing was performed as shown in the lower panel. (C) Isolated fractions were detected by immunoblotting. (D) Localization of SERPINB1 in YTS cells was visualized via confocal microscopy. GzmH, perforin, LAMP1, and EEA1 staining red (left panels), SERPINB1 staining green (middle panels), and merged images (right panels). Original magnification 3400. These images are representative of at least four separate experiments. 1322 SERPINB1 INHIBITS THE ACTIVITY OF HUMAN GRANZYME H refinement statistics are given in Table I. Stereochemical parameters were of GzmH, we isolated secretory lysosomes of YTS cells using evaluated with PROCHECK (18). Figures showing structural representa- self-forming Percoll gradients (Fig. 1A). Based on the size and tions were prepared with the program PyMOL (http://www.pymol.org/). high density, secretory lysosomes could be enriched after frac- Structure modeling of GzmH with SERPINB1 tionation. To separate the soluble contents from membrane-asso- ciated proteins, the pellet granules were subjected to freeze–thaw The initial models were constructed with Modeler software (19), using the ∼ serpin–trypsin complex (PDB code 1K9O) (20) as the Michaelis complex cycles prior to SDS-PAGE resolution. Three major proteins ( 66, model for the SERPINB1–GzmH complex and the a1PI/PPE covalent 45, and 35 kDa) were visualized in the supernatants by Coomassie complex (PDB code 2D26) (21) for the inhibitory complex. The active site staining (Fig. 1B). Each band was cut and digested by trypsin for was refined by our previously solved GzmH–decapeptide complex (PDB peptide sequencing. The first band ∼66 kDa primarily contained code 3TJV) (10). In the inhibitory complex model, the carbon atom of the ∼ carbonyl group of Phe343 in serpin is linked to the oxygen atom of the HSP70 and perforin. The second band 45 kDa included SER- Ser197 side chain via a single bond. The all-atomic Molecular Dynamics PINB1, SERPINB8, and SERPINB9. The bottom band ∼35 kDa simulations were performed for both models to relax them and eliminate contained GzmB (56.7%) and GzmH (14.6%), which is consistent local unreasonable conformation caused by residue mutation or artificial with previous reports (3, 24). Notably, much greater amounts of effect (22, 23). The last snapshots of 50-ns Molecular Dynamics trajec- SERPINB1 (32.4%) were contained in the second band compared tories were extracted for structural analysis. with SERPINB9 or SERPINB8. We observed that SERPINB1 was distributed in the cytoplasmic sections and also fractionated with Results GzmH in the purified granules (F19–F24), as well as in the early SERPINB1 is fractionated with GzmH from the separation of endosomes (F10–F13) (Fig. 1C). EEA1 (early endosome marker) NK cell granules was used to distinguish between early endosomes and lysosomes. The human NK cell line YTS is well established as a main expres- SERPINB1 was distributed in the cytosol and partially colocalized Downloaded from sion cell line of GzmH (3, 10). To identify intracellular inhibitors with GzmH in YTS cells, as observed by confocal microscopy

FIGURE 2. SERPINB1 associates with GzmH to form an SDS-stable serpin–protease complex. (A) wtSERPINB1 can pull down GzmH. His-tagged http://www.jimmunol.org/ wtSERPINB1 attached to Ni-NTA beads was incu- bated with purified D102N-GzmH at 4˚C for 2 h. Binding proteins were visualized by immunoblotting with anti-GzmH Ab (upper panel) or anti-SERPINB1 Ab (lower panel). The naked beads served as a non- specific control. (B) GzmH binds to SERPINB1. D102N-GzmH conjugated to Affi-Gel10 beads was incubated with purified SERPINB1 at RT for 30 min, followed by immunoblotting. (C) SERPINB1 forms by guest on October 1, 2021 a Michaelis complex with inactive GzmH. A mixture of purified wtSERPINB1 and D102N-GzmH proteins was incubated for 5 h at 4˚C at a molar ratio of 1:2, or the individual protein was injected separately into a Superdex 75 column running with 40 mM Tris (pH 8), 150 mM NaCl. The elution fractions of the mix- ture (gray line) and individual proteins (D102N- GzmH, dashed line; SERPINB1, black line) were recorded with a flow rate of 0.5 ml/min and collected as 0.5-ml fractions after injection into the column in the upper panel. Proteins in the elution were visual- ized by Coomassie Brilliant Blue staining after res- olution. D102N-GzmH (26 kDa) showed the protein peak at 13.8 ml and SERPINB1 (45 kDa) at 11.65 ml. The mixture showed an earlier elution volume at 10.8 ml, which contained both SERPINB1 and D102N- GzmH proteins. The latter peak of the mixture con- tained excessive D102N-GzmH. (D)SERPINB1 forms an SDS-stable inhibitory complex with active GzmH. Aliquots of SERPINB1 (0.5 mM) were trea- ted separately with active Gzm (0.5 mM) and then subjected to SDS-PAGE. The black arrow denotes SERPINB1, the white arrow marks Gzm, and the gray arrow points to the SDS-stable complex formed by SERPINB1 and Gzm. (E) SERPINB8 fails to form an SDS-stable serpin–protease complex with active GzmH. Aliquots of SERPINB8 were incubated sep- arately with all five Gzm at 37˚C and visualized by Coomassie Brilliant Blue staining. The black arrow represents the individual Gzm, the open arrow SER- PINB1 or SERPINB8, and the gray arrow covalent Gzm-inhibitor complexes. The Journal of Immunology 1323

(Fig. 1D). SERPINB1 localized with a lysosome marker LAMP1, mixture or aliquots of individual protein were injected into a which indicates that SERPINB1 is also localized in the granules Superdex 75 column for size-exclusion chromatography (Fig. 2C, known as specialized secretary lysosomes. Because SERPINB1 upper panel). Elution fractions were resolved with SDS-PAGE, is a typical clade B serpin molecule, it does have a membrane- followed by Coomassie staining (Fig. 2C, lower panel). We ob- spanning domain, suggesting that it is a cytosolic molecule. How served that wtSERPINB1 and D102N-GzmH both appeared in SERPINB1 functions in NK cell granules requires further in- a higher molecular mass peak of 70 kDa than did wtSERPINB1 vestigation. molecule alone (45 kDa) or D102N-GzmH alone (26 kDa). The molecular masses were calculated by calibration standards. SERPINB1 associates with GzmH to form a classical Serpins are known as suicide inhibitors; they first bind to a spe- SDS-stable serpin–protease complex cific target protease to form a Michaelis complex. Then, the serpin We wanted to determine the interaction between SERPINB1 and forms an SDS-stable covalent complex with the target protease upon GzmH. Soluble His-tagged wtSERPINB1 was expressed and pu- cleavage by this protease to block enzymatic activity (20). To test rified from E. coli. We refolded tag-free active human GzmH whether SERPINB1 forms an SDS-stable covalent-bound complex (wtGzmH) and enzymatically inactive D102N-GzmH from in- with GzmH, we incubated wtSERPINB1 with wtGzmH (Fig. 2D). clusion bodies, as previously described (10). wtGzmH showed Notably, an SDS-stable complex band was observed as the result proteolytic activity, whereas D102N-GzmH had no activity for of the incubation of wtSERPINB1 with wtGzmH, whereas weak the synthetic substrate Ac-PTSY-pNA. D102N-GzmH was incu- complex bands were also observed in the incubation with GzmB and 2+ bated with SERPINB1-bound Ni resin or Ni resin alone at 4˚C GzmM. In contrast, inactive D102N-GzmH had no such activity. for 2 h. His-SERPINB1 beads efficiently pulled down D102N- However, SERPINB8, another serpin molecule found in the cyto- GzmH (Fig. 2A). D102N-GzmH–immobilized Affi-Gel 10 beads toxic granules, failed to interact with GzmH or other Gzm (Fig. 2E). Downloaded from were also able to bind wtSERPINB1 (Fig. 2B). The naked beads were used as a negative binding control. To further verify the SERPINB1 directly blocks the proteolytic activity of GzmH binding affinity of SERPINB1 for GzmH, we mixed wtSERPINB1 To determine whether SERPINB1 can inhibit the enzymatic ac- with D102N-GzmH at a molar ratio of 1:2 on ice for 5 h, and the tivity of GzmH, we measured the concentrations of wtGzmH and http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. SERPINB1 represses the enzymatic activity of GzmH. (A) SERPINB1 inhibits the activity of GzmH. Gzm were incubated with SERPINB1 at different molar ratios at 37˚C for 1 h. Residual activities of Gzm were measured as described in Materials and Methods. Data are mean 6 SD and are representative of five independent experiments. (B) SERPINB1 suppresses GzmH-induced substrate cleavage. 721.221 cell lysates (1 3 105 equivalents) were incubated with active GzmH (wtGzmH) and increased doses of SERPINB1. All samples were analyzed by SDS-PAGE and probed with the indicated Abs. NM23-H1 was detected as a loading control. (C) wtSERPINB1 forms an SDS-stable complex with wtGzmH in a dose-dependent manner. Aliquots of wtGzmH were incubated with different concentrations of wtSERPINB1 at the indicated molar ratios at 37˚C for 1 h, followed by Coomassie Brilliant Blue staining. D102N-GzmH was not trapped by SERPINB1 and was used as a negative control. (D) wtGzmH and wtSERPINB1 form an SDS-stable complex in a time-dependent manner. wtSERPINB1 (0.5 mM) and wtGzmH (0.5 mM) were incubated for different times at 37˚C. GzmH inhibitor was used as a negative control. The black arrows denote SERPINB1, the white arrows mark GzmH, the gray arrows mark the SDS-stable complex, the black arrowheads denote the cleaved fragments of La, and the open arrowhead marks cleaved bands of ICAD. All data are representative of at least three separate experiments. 1324 SERPINB1 INHIBITS THE ACTIVITY OF HUMAN GRANZYME H wtSERPINB1 and then incubated aliquots of wtGzmH with dif- the activity of wtGzmH. Taken together, these results indicate that ferent doses of wtSERPINB1, followed by examination of enzy- SERPINB1 disables the enzymatic activity of GzmH through the matic activity. We found that SERPINB1 inhibited the proteolytic covalent complex formation. 6 activity of GzmH with a stoichiometry of inhibition (SI) of 1.55 343 0.27 and an apparent second-order rate constant of 1.10 (6 0.12) 3 GzmH cleaves SERPINB1 at Phe in the RCL 105 M21s21 (Fig. 3A, Supplemental Table I). However, SER- SERPINB1 was reported to inhibit elastase- and chymotrypsin-like PINB1 failed to suppress the enzymatic activities of the other four proteases through degradation of its RCL (25). GzmH is a chy- granzymes (Fig. 3A), which is in agreement with a previous report motrypsin-like protease with preference for bulky residues, such (25). To assess whether SERPINB1 inhibition is physiologically as Tyr and Phe, at the P1 position (27). As shown in Fig. 4A, only relevant, we incubated wtGzmH with tumor cell lysates by adding one Phe residue, Phe343, is present in the RCL. To determine the increasing concentrations of wtSERPINB1 (Fig. 3B). As expected, cleavage site of SERPINB1 by GzmH, we mutated Phe343 to Ala wtSERPINB1 suppressed the cleavage of known physiological (F343A-SERPINB1) and generated its recombinant protein. In- substrates, including La and ICAD. At a concentration of 1 mM, terestingly, F343A-SERPINB1 lost its inhibitory action against wtSERPINB1 completely blocked the proteolysis of both sub- GzmH (Fig. 4B, middle panel), whereas wtSERPINB1 exhibited strates. The GzmH synthetic inhibitor Ac-PTSY-CMK was able to strong inhibition activity (Fig. 4B, left panel). Additionally, F343A- inhibit the enzymatic activity of GzmH (Fig. 3B), which was used SERPINB1 failed to form the stable SERPINB1–GzmH com- as an inhibition control. Gzm A–activated endonuclease NM23-H1 plex (Fig. 4C), whereas wtSERPINB1 formed the complex with in the SET complex (26) was unable to be cleaved by GzmH (10), wtGzmH. which was used as a good loading control. We (10) and other investigators (8) demonstrated that acidic To further confirm that SERPINB1 covalently binds to GzmH, residues in the P39 and P49 sites of physiological substrates are Downloaded from we incubated wtGzmH with wtSERPINB1 at different molar ratios. preferred for human GzmH. wtSERPINB1 bears Leu346 and Met347 Expectedly, a stable SERPINB1–GzmH inhibitory complex was at the P39 and P49 sites, respectively. Thus, we mutated both Leu346 formed with the predicted molecular mass of ∼70 kDa in a dose- and Met347 to Asp (LM-DD-SERPINB1), as shown in Fig. 4A. dependent fashion (Fig. 3C). Inactive D102N-GzmH mutant failed LM-DD-SERPINB1 exhibited a stronger inhibitory effect on pro- to form such a covalent complex, suggesting that the formation of teolysis of GzmH than did wtSERPINB1 (Fig. 4B, right panel). In the stable SERPINB1–GzmH complex requires the enzymatic addition, the SI for LM-DD-SERPINB1 to GzmH was close to 1, http://www.jimmunol.org/ activity of GzmH. This stable SERPINB1–GzmH complex also indicating that LM-DD-SERPINB1 augments the efficiency of inhi- appeared in a time-dependent manner (Fig. 3D). The Ac-PTSY- bition. We also calculated the association rate constant for GzmH CMK inhibitor abrogated the complex formation by inhibition of inhibition in the presence of wtSERPINB1 or LM-DD-SERPINB1 by guest on October 1, 2021 FIGURE 4. Inhibition of GzmH by SERPINB1 occurs through cleavage of the RCL. (A) Schematic diagram showing the recognition modes of GzmH against SERPINB1. The P1 residue for GzmH was predicted to be Phe343. The putative acting P1 residue Phe343 wasmutatedtoAlaasF343A- SERPINB1, whereas the P29 P39 residues Leu346– Met347 were mutated into Asp346–Asp347 as LM- DD-SERPINB1. (B) Phe343A mutation abolishes its inhibitory action to GzmH. Increasing concen- trations of SERPINB1 variants were incubated with wtGzmH (100 nM) at 37˚C for 2 h. Then synthetic substrates were added, and remaining activity was measured. The SI was calculated by linear regres- sion analysis. (C) F343A-SERPINB1 mutant fails to form the covalent complex. F343A-SERPINB1 and wtSERPINB1 were incubated with wtGzmH at 37˚C for 1 h, followed by Coomassie brilliant blue staining (upper panel) and immunoblotting against SERPINB1 (middle panel) or GzmH (lower panel). (D) The LM-DD-SERPINB1 variant promotes the stable serpin–protease complex formation. LM- DD-SERPINB1 and wtSERPINB1 were incubated with wtGzmH at 37˚C for 1 h, followed by Coo- massie staining. The black arrows denote SER- PINB1, the white arrows mark GzmH, and the gray arrows mark the SDS-stable complex. WB, Im- munoblotting. The Journal of Immunology 1325

(Supplemental Table I). We found that LM-DD-SERPINB1 enhanced lysates were incubated at 4 or 37˚C for the indicated times. As the inhibition efficiency by 5-fold, with an apparent second-order shown in Fig. 5A, the stable covalent complex was generated at rate constant of 5.37 (6 0.60) 6 105 M21s21. LM-DD-SERPINB1 37˚C under which conditions GzmH recovered its enzymatic also strengthened the formation of the stable SERPINB1–GzmH activity. The Ac-PTSY-CMK inhibitor abolished the formation complex (Fig. 4D). Collectively, these results identify that Phe343 of the stable covalent complex (Fig. 5B). Addition of wtGzmH in the RCL is the P1 site for GzmH. produced a stable covalent complex band that was lower than that of the native GzmH, largely due to glycosylation of native Overexpression of SERPINB1 suppresses GzmH- or LAK GzmH in YTS cells (4). Knockdown of SERPINB1 by siRNA cell–mediated cell death (Fig. 5C) or knockdown of GzmH by short hairpin RNA (Fig. Given that SERPINB1 was initially isolated from YTS cells, we 5D) attenuated the formation of the stable complex. Notably, the explored whether native SERPINB1 can inhibit native GzmH classical SDS-stable protease inhibitor complex was observed when they encounter each other in cells. Because GzmH is stored in primary NK cells (Fig. 5E). However, SERPINB1 could not in the acidic granules, we disrupted all of the membrane com- form a covalent complex with inactive GzmH in the lysates of partments using a buffer containing 0.5% Nonidet P-40. YTS cell YTS and primary NK cells at 4˚C (Fig. 5A, 5E), indicating that Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. SERPINB1 overexpression inhibits GzmH- and LAK cell–mediated cell death. (A) The SDS-stable SERPINB1–GzmH complex is formed in lysates of YTS cells. Lysates of YTS cells were incubated for different times at 37˚C or at 4˚C for 2 h, followed by immunoblotting. The SERPINB1–GzmH complex (∼70 kDa) was formed only when cell lysates were incubated at 37˚C. (B) GzmH inhibitor abolishes the complex formation. wtGzmH or the synthetic GzmH inhibitor (Ac-PTSY-CMK; inh) was added to YTS cell lysates and detected as in (A). Silencing of SERPINB1 (C) or GzmH (D) reduces the complex formation. SERPINB1 and GzmH were knocked down separately in YTS cells by siRNA or short hairpin RNA, and treated as above. In lanes 1 and 4, lysates were incubated at 4˚C for 2 h; in lanes 2 and 5, lysates were incubated at 4˚C for 1 h and at 37˚C for 1 h; and in lanes 3 and 6, lysates were incubated at 37˚C for 2 h. (E) The complex exists in primary NK cells. Primary NK cell lysates were incubated for the indicated times at 37 or 4˚C and treated as above. The asterisk marks a nonspecific band of GzmH. (F) Jurkat cell lines with wtSERPINB1 and F343A-SERPINB1 expression were established. Jurkat cells were infected with lentivirus encoding wtSERPINB1 or F343A-SERPINB1, and stable cell lines were selected by puromycin. wtSERPINB1 and F343A-SERPINB1 were examined by Western blot. b-actin served as a loading control. (G) Overexpression of wtSERPINB1 represses GzmH-induced cell death. Jurkat cells stably overexpressing empty vector, wtSERPINB1, or F343A-SERPINB1 were treated with 1 mM GzmH (left panel) or 1 mM GzmB (right panel) plus adenovirus (Ad) at 37˚C for 4 h, followed by flow cytometry. Total dead cells, including Annexin V and propidium iodide single-positive and double-positive cells, are shown as mean 6 SD. (H) Overexpression of wtSERPINB1 protects Jurkat cells from LAK cell–mediated cytolysis. Jurkat cells overexpressing empty vector, wtSERPINB1, or F343A-SERPINB1 were labeled with 51Cr and incubated with IL-2–activated LAK cells at an E:T ratio of 1. For a specific inhibition assay, LAK cells were pretreated with 500 nM CMA for 2 h or with 300 mM GzmH inhibitor for 1 h before incubation with target cells. **p , 0.01. The gray arrows mark the SDS-stable complex formed by endogenous SERPINB1 and GzmH; the black arrow denotes the complex formed by endogenous SERPINB1 and recombinant wtGzmH. All data are representative of at least three separate experiments. n.s., Not significant. 1326 SERPINB1 INHIBITS THE ACTIVITY OF HUMAN GRANZYME H

GzmH is not initially covalent in NK cells. The proteolytic Overall structures of SERPINB1 and active GzmH activities of all granzymes are inactive in acidic pH granules Although wtSERPINB1 failed to crystallize, we were able to de- (1); therefore, GzmH should remain inactive in the granules and termine the crystal structure of the LM-DD-SERPINB1 mutant at be unable to cleave SERPINB1 to form a covalent inhibition 2.9-A˚ resolution (Table I). The SERPINB1 molecule folds into complex. a typical serpin architecture composed of three large b-sheets and We next determined whether SERPINB1 inhibits the proteolytic nine a-helices. The two molecules of LM-DD-SERPINB1 in the activity of GzmH in cells overexpressing SERPINB1. wtSER- asymmetric unit adopt a conformation known as the native or PINB1 or F343A-SERPINB1 mutant was overexpressed in Jurkat stressed state of the serpin (11), in which the RCL is flexibly exposed cells, and their stable cell lines were established (Fig. 5F). These and only five strands exist in the A sheet (Supplemental Fig. 1E). transfected Jurkat cells were loaded with wtGzmH with adeno- The two LM-DD-SERPINB1 molecules could be assembled virus. wtSERPINB1 overexpression dramatically suppressed into two types of asymmetric units. The first one is that the two GzmH-induced apoptosis compared with the empty vector con- monomeric molecules stand upside down (Supplemental Fig. 1A). trol (Fig. 5G). However, F343A-SERPINB1 transfection did not However, in addition to the main chain orientation, the difference suppress cell death compared with the empty vector control (Fig. density map of chain A could always be traced to the symmetric 5G, left panel). However, wtSERPINB1 overexpression failed to molecule of chain B at the converged position of Cys344 (Sup- inhibit GzmB-mediated cytotoxicity (Fig. 5G, right panel). PBMCs plemental Fig. 1B). Therefore, we adopted the second asymmetric were incubated with IL-2 to produce LAK cells. The transfected unit, in which two LM-DD-SERPINB1 molecules are linked by Jurkat cells were incubated with LAK cells at an E:T ratio of 1. As a disulfide bridge (Fig. 6A). Cys344 at the P19 position in the RCL expected, wtSERPINB1 transfection remarkably suppressed LAK mediates the disulfide bond formation. RCL normally contains 17 Downloaded from cell–mediated cytolysis compared with the empty vector control residues and is tethered between b-sheets A and C (28). In our (Fig. 5H). F343A-SERPINB1 overexpression almost recovered structure, residues 332–341 in chain A and 330–341 in chain B are the cytotoxic capacity of LAK cells. Moreover, CMA, an inhib- disordered because they lack clear electron density. Residues 342– itor of cytotoxic exocytosis, was mostly able to suppress LAK 349 [from P2 to P69, nomenclature described by Schechter and cell–mediated cell death (Fig. 5H). Additionally, the Ac-PTSY- Berger (29)] are visible with well-defined electron density, as CMK inhibitor markedly reduced LAK cell–mediated cytolysis, shown in Supplemental Fig. 1B. Other than the disulfide bond, http://www.jimmunol.org/ but it still retained near 20% cytotoxicity, which suggests that there is little contact between the two LM-DD-SERPINB1 mole- other Gzm in addition to GzmH participate in the induction of cules (Fig. 6A); therefore, this dimer is unlikely to be physiolog- cytolysis. ically relevant. Actually, we did not observe any dimer formation

Table I. X-ray diffraction data collection and refinement statistics

Parameter SERPINB1 wtGzmH by guest on October 1, 2021 Data collection Space group P41 P21212 Cell dimensions a, b, c (A˚ ) 67.3, 67.3, 180.3 170.5, 366.8, 61.2 a, b, g (˚) 90, 90, 90 90, 90, 90 Resolution (A˚ ) 50–2.9 (3.0–2.9) 40–3.0 (3.1–3.0) a Rmerge 0.079 (0.439) 0.082 (0.314) I/s(I) 35.0 (7.0) 19.7 (6.3) Completeness (%) 100 (99.9) 100 (100) Redundancy 15.1 (14.7) 7.0 (7.1) Wilson plot B factor 77.4 58.1 Refinement Resolution (A˚ ) 50–2.9 40–3.0 No. reflections 16,846 74,302 b Rwork/Rfree 0.183/0.232 0.271/0.306 No. atoms Protein 5,893 21,176 Water 17 38 Ion 78 B factors Protein 74.8 70.1 Water 67.1 62.1 Ion 67.8 Total 74.8 70.1 Root mean square deviations Bond length (A˚ ) 0.006 0.012 Bond angles (˚) 1.12 1.86 Ramachandran plot Most favored (%) 87.0 75.2 Additionally allowed (%) 12.7 23.9 Generously allowed (%) 0.3 0.9 Disallowed 0 0 Highest-resolution shell statistics are shown in parentheses. a Rmerge is defined by Rmerge = ShSi |Iih 2,Ih. |/ShSi ,Ih., where ,Ih. is the mean of the observations Iih of reflection h. b Rwork is defined by Rwork = S(||Fp(obs)| 2 |Fp(calc)||)/ S|Fp (obs)|; Rfree = R factor for a selected subset (5%) of the reflections that was not included in prior refinement calculations. The Journal of Immunology 1327 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Overall structures of SERPINB1 and active GzmH, as well as the distorted suicide mechanism. (A) Overall structure of LM-DD-SERPINB1 variant. The three b-sheets and nine helices (hA–hI; cyan) are highlighted. The exposed RCL is shown in magenta, and the missing residues in RCL are denoted as dotted lines. Cys344, which mediates the disulfide bond, is depicted as spheres. (B) Twelve molecules of wtGzmH in (Figure legend continues) 1328 SERPINB1 INHIBITS THE ACTIVITY OF HUMAN GRANZYME H in YTS cell lysates or isolated granules (Supplemental Fig. 1C, To coordinate with the dramatic conformational changes, GzmH is 1D). distorted and inactivated. The catalytic triad is disordered to move We previously reported the crystal structures of inactive GzmH Ser195 ∼5A˚ away from its catalytic partner His57 (Fig. 6E), which mutant (D102N-GzmH) (10). In this study, we solved the crystal is consistent with previous reports (20, 21). structure of enzymatically active GzmH (wtGzmH). Interestingly, the 12 wtGzmH molecules in the asymmetric unit are assembled Discussion into two six-petal rings (Fig. 6B). wtGzmH is a monomer in so- Gzm-mediated cytotoxicity plays a critical role in immune re- lution, and this six-petal ring is probably not physiologically rel- sponses against infections and tumors. However, the balance be- evant. All 12 chains adopt the classical serine protease fold with tween activation and inhibition of the proteolytic cascade must be similar rigid structures. RMS distances among equivalent Ca tightly modulated to avoid self damage. In a recent study, we atoms of the 12 molecules are no more than 1.0 A˚ , suggesting that crystallized the enzymatically inactive D102N-GzmH alone and in they are identical structures overall (Supplemental Fig. 2A). There complex with its synthetic substrate and inhibitor and solved their are recognizable differences in the conformation of several of the crystal structures (10). We defined the mechanisms of substrate loops on the surface, away from the active site. We superimposed recognition and enzymatic activation. In this study, we identified wtGzmH with D102N-GzmH alone and its complex with sub- SERPINB1 as a potent intracellular inhibitor for human GzmH. strate. As shown in Supplemental Fig. 2B, wtGzmH remains Upon cleavage of the RCL at Phe343, SERPINB1 formed an SDS- nearly unchanged with respect to its substrate binding, suggesting stable covalent complex with GzmH in YTS and primary NK that the substrate catalysis of GzmH involves little conformational cells. Overexpression of SERPINB1 suppressed GzmH- or LAK change. cell–mediated cell death. We tried for years to crystallize human Downloaded from active GzmH and full-length SERPINB1 and failed to obtain the SERPINB1 inhibits the catalytic activity of GzmH by complex structure. Through molecular modeling, SERPINB1 in- deformation hibits GzmH by deformation, which is reminiscent of other serpin– Although the structures of several serpin–protease complexes were protease complexes. reported (30, 31), only two crystal structures of the inhibitory Cytotoxic granules are specialized secretory lysosomes in NK complexes have been solved (20, 21). Despite extensive efforts, cells. Because of their high density, it was possible to separate we were unable to cocrystallize SERPINB1 with wtGzmH. Thus, secretory lysosomes by self-forming Percoll gradients (24). In the http://www.jimmunol.org/ we modeled the binary Michaelis complex and the inhibitory isolated supernatants, three serpin family members SERPINB1/ complex by docking the crystal structure of SERPINB1 to SERPINB8/SERPINB9 were present in NK cells in addition to wtGzmH (Fig. 6C). In the native state, the RCL of SERPINB1 the granule components perforin, GzmB, and GzmH. SERPINB9 (especially Phe343) is exposed to the solvent. Upon encountering was reported to be a physiological inhibitor for human GzmB GzmH, RCL is inserted into the active site groove as a substrate. (12), which protects lymphocytes from GzmB-induced injury. A Phe343 (P1) fits nicely into the hydrophobic cave of the S1 pocket recent study showed that SERPINB4 is an intracellular inhibitor (Fig. 6D). Additionally, beneath the S1 pocket, Lys192 forms a for human GzmM that suppresses GzmM-mediated cell death 335

strong hydrogen bond with the carbonyl group of Ala in (13). We showed that SERPINB1 and SERPINB8 resided in YTS by guest on October 1, 2021 SERPINB1, which enhances the interaction. The RKR motif of cells; however, it is unknown whether they inhibit Gzm. GzmH (10) interacts tightly with the Asp346 and Asp347 residues. SERPINB1, also known as monocyte/ elastase in- Three salt bridges, one between Lys40 (GzmH) to Asp346 (SER- hibitor, was identified in human monocytes/ to act as a PINB1) and another two between Arg39 (GzmH), Arg41 (GzmH) specific and efficient inhibitor for elastase, , and pro- to Asp347 (SERPINB1), respectively, were also identified. Inter- teinase-3 (32). SERPINB1 is highly expressed in the cytosol of estingly, Lys74 protrudes into the RKR motif and forms a hydro- neutrophils and prevents infection-induced tissue injury (33). gen bond with Asp347. Except for the electrostatic interactions as Moreover, SERPINB1 can maintain a healthy neutrophil reserve mentioned above, the Nh1 atom of Arg39 forms a hydrogen bond in the bone marrow (34), suggesting that it plays a crucial role in with the carbonyl group of Pro348. We deduced that wtSERPINB1 neutrophil development and homeostasis. We demonstrated that only maintains the main chain interactions with the RKR motif SERPINB1 also resides in NK cells, and its overexpression sup- and, thus, lacks those electrostatic interactions. This may explain presses GzmH- or LAK cell–mediated cytolysis. Notably, SER- why LM-DD-SERPINB1 augments the inhibition efficiency PINB1 forms a canonical SDS-stable serpin–protease complex with against GzmH than wtSERPINB1. both recombinant and native forms of GzmH. These data suggest Once the RCL inserts into the binding sites of GzmH, the that SERPINB1 inhibits GzmH through a conformational-change catalytic triad is activated, and Ser195 of GzmH attacks the peptide suicide mechanism. bond between Phe343 and Cys344 of SERPINB1. After acylation of Based on our structural superimpositions, the overall structures Ser195, the covalent complex forms by pulling GzmH by the RCL and steric positions of active centers of active GzmH and its mutant from one pole to another to generate a new b-sheet S4A (Fig. 6C). D102N-GzmH are similar. However, active GzmH was crystallized

an asymmetric unit. Chains are labeled from “a” to “l.” (C) A full view of the interaction between SERPINB1 and GzmH. Binary Michaelis complex and inhibitory complex formed by GzmH and SERPINB1 were generated by docking the structures of SERPINB1 to GzmH. SERPINB1 molecules are shown in cyan with central b-sheets in yellow and RCL in magenta. Phe343 residue in SERPINB1 is shown as magenta balls and sticks. GzmH (chain B) is green, and the catalytic triad residues (His57, Asp102, and Ser195) are violet. (D) Interaction of SERPINB1 RCL with GzmH in the active site cleft in the Michaelis complex. Total structure of GzmH is shown as electrostatic surface, and residues that form the S and S9 pockets are represented by green sticks. Residues of RCL from 339 to 348 (GIATFCMDDP) are shown as magenta balls and sticks. Nitrogen atoms are blue, and oxygen atoms are red. Lower panel, RKR motif interactions, showing the salt bridges and hydrogen bonds formation. (E) Conformational distortions of GzmH encounters SERPINB1. The GzmH chain in the native state (cyan) was superimposed to GzmH in the Michaelis complex (green) and inhibitory complex (purple) on the Ca atoms. The superimposition result is shown as cartoons (lower panel) in the standard orientation (10). Distortion of the active triad is outlined by the smaller red box and enlarged in the larger red box. The red arrows indicate the direction of loop shifts. The Journal of Immunology 1329 with 12 molecules in the asymmetric unit, which might be caused 3. Fellows, E., S. Gil-Parrado, D. E. Jenne, and F. C. Kurschus. 2007. Natural killer cell-derived human granzyme H induces an alternative, caspase-independent by crystallographic packing. It is distinct from D102N-GzmH (10). cell-death program. Blood 110: 544–552. Actually, GzmH exists as a monomer in solutions and in cells. 4. Sedelies, K. A., T. J. Sayers, K. M. Edwards, W. Chen, D. G. Pellicci, Interestingly, the crystal of LM-DD-SERPINB1 also contains two D. I. Godfrey, and J. A. Trapani. 2004. Discordant regulation of granzyme H and granzyme B expression in human lymphocytes. J. Biol. Chem. 279: 26581–26587. molecules linked by a disulfide bond in the asymmetric unit. Both 5. Waterhouse, N. J., and J. A. Trapani. 2007. H is for helper: granzyme H helps active GzmH and SERPINB1 exist as a monomer in NK cell lysates. granzyme B kill adenovirus-infected cells. Trends Immunol. 28: 373–375. Indeed, formation of the serpin–protease complex requires each 6. Hou, Q., T. Zhao, H. Zhang, H. Lu, Q. Zhang, L. Sun, and Z. Fan. 2008. Granzyme H induces apoptosis of target tumor cells characterized by DNA fragmentation and monomeric form. Bid-dependent mitochondrial damage. Mol. Immunol. 45: 1044–1055. GzmH needs bulky aromatic amino acids to compensate for its 7. Andrade, F., E. Fellows, D. E. Jenne, A. Rosen, and C. S. Young. 2007. Gran- 343 zyme H destroys the function of critical adenoviral proteins required for viral hydrophobic cavity at the S1 position. SERPINB1 harbors Phe DNA replication and granzyme B inhibition. EMBO J. 26: 2148–2157. in the RCL, consistent with the catalytic specificity preference of 8. Romero, V., E. Fellows, D. E. Jenne, and F. Andrade. 2009. Cleavage of La GzmH. Mutation of Phe343 to Ala completely abolishes the in- protein by granzyme H induces cytoplasmic translocation and interferes with La- mediated HCV-IRES translational activity. Cell Death Differ. 16: 340–348. hibitory effect of SERPINB1, as well as the covalent complex 9. Tang, H., C. Li, L. Wang, H. Zhang, and Z. Fan. 2012. Granzyme H of formation. We concluded that Phe343 is the acting P1 residue for cytotoxic lymphocytes is required for clearance of the hepatitis B virus through GzmH cleavage. Actually, Phe343 locates at the P2 position of cleavage of the hepatitis B virus X protein. J. Immunol. 188: 824–831. 10. Wang, L., K. Zhang, L. Wu, S. Liu, H. Zhang, Q. Zhou, L. Tong, F. Sun, and RCL, effectively shortening the RCL by one residue relative to Z. Fan. 2012. Structural insights into the substrate specificity of human gran- most serpins. Other chymotrypsin-like proteases, such as cathep- zyme H: the functional roles of a novel RKR motif. J. Immunol. 188: 765–773. 11. Huntington, J. A. 2011. Serpin structure, function and dysfunction. J. Thromb. sin G, chymase, and chymotrypsin, were also reported to interact Haemost. 9(Suppl. 1): 26–34. 343 with SERPINB1 via Phe (25), indicating that a similar inhibi- 12. Zhang, M., S. M. Park, Y. Wang, R. Shah, N. Liu, A. E. Murmann, C. R. Wang, tion mechanism was adopted by SERPINB1. The acting P19 site M. E. Peter, and P. G. Ashton-Rickardt. 2006. Serine protease inhibitor 6 protects Downloaded from 344 cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic (actually the P1 site of RCL) residue Cys was identified to granules. Immunity 24: 451–461. inactivate the (25). Although Cys344 mediates 13. de Koning, P. J., J. A. Kummer, S. A. de Poot, R. Quadir, R. Broekhuizen, the disulfide bridge formation in our crystal structure, no dimeric A. F. McGettrick, W. J. Higgins, B. Devreese, D. M. Worrall, and N. Bovenschen. 2011. Intracellular serine protease inhibitor SERPINB4 inhibits granzyme molecules were detected in NK cells. Therefore, we defined the M-induced cell death. PLoS ONE 6: e22645. SERPINB1 molecule as a disulfide bond–tethered monomer in the 14. Hua, G., Q. Zhang, and Z. Fan. 2007. Heat shock protein 75 (TRAP1) antago-

nizes reactive oxygen species generation and protects cells from granzyme M- http://www.jimmunol.org/ crystal structure. GzmH prefers P39 and P49 acidic residues, as we mediated apoptosis. J. Biol. Chem. 282: 20553–20560. (8) and other investigators (10) reported. We modified the hy- 15. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data col- drophobic residues Leu346 and Met347 to Asps at the P39 and P49 lected in oscillation mode. Methods Enzymol. 276: 307–326. 16. Read, R. J. 2001. Pushing the boundaries of molecular replacement with max- positions and demonstrated that the double mutations of SER- imum likelihood. Acta Crystallogr. D Biol. Crystallogr. 57: 1373–1382. PINB1 led to improved inhibitory effect. Structural modeling 17. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular provides further insights into the SERPINB1 binding and inhibi- graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126–2132. 18. Laskowski, R. A., J. A. Rullmannn, M. W. MacArthur, R. Kaptein, and tory mechanism for GzmH. J. M. Thornton. 1996. AQUA and PROCHECK-NMR: programs for checking Serpin family members play pivotal roles in the inhibition of the quality of protein structures solved by NMR. J. Biomol. NMR 8: 477–486. serine proteases in blood coagulation, mental diseases, and innate 19. Eswar, N., B. Webb, M. A. Marti-Renom, M. S. Madhusudhan, D. Eramian, M.-y. Shen, U. Pieper, and A. Sali. 2006. Comparative protein structure mod- by guest on October 1, 2021 immune responses. SERPINB1 was first identified as a fast-acting eling using Modeller. Curr. Protoc. Bioinformatics 15: 5.6.1–5.6.30. inhibitor produced in monocytes and neutrophils (35). Because of 20. Huntington, J. A., R. J. Read, and R. W. Carrell. 2000. Structure of a serpin- protease complex shows inhibition by deformation. Nature 407: 923–926. the lack of a cleavable hydrophobic signal sequence, SERPINB1 21. Dementiev, A., J. Dobo´, and P. G. Gettins. 2006. 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Increasing California San Francisco, San Francisco, CA. Available at: http://ambermd.org/. evidence shows that GzmH participates in the defense of inflam- 24. Casey, T. M., J. L. Meade, and E. W. Hewitt. 2007. Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell mation (7–9). Thus, SERPINB1 might be involved in the protec- secretory lysosome. Mol. Cell. Proteomics 6: 767–780. tion of healthy NK cells, as well as in NK cell–mediated tissue 25. Cooley, J., T. K. Takayama, S. D. Shapiro, N. M. Schechter, and E. Remold- injury during inflammation. O’Donnell. 2001. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry 40: 15762–15770. Acknowledgments 26. Fan, Z., P. J. Beresford, D. Y. Oh, D. Zhang, and J. Lieberman. 2003. Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated We thank Dr. Baoxue Ge for providing the YTS and 721.221 cell lines and apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 112: Dr. R.P. Sekaly for providing the Ab for ICAD. We also thank Zhensheng 659–672. Xie of the Institute of Biophysics core facility for helping with mass 27. Edwards, K. M., C. M. Kam, J. C. Powers, and J. A. Trapani. 1999. The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like spectrometry; Qiangjun Zhou and Jun Ma for assistance with data collec- (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of tion; and Chong Li, Shuo Wang, Haidong Tang, Jun Chen, and Jun Zhou granzyme B-containing endosomes. J. Biol. Chem. 274: 30468–30473. for critical discussions. 28. Silverman, G. A., P. I. Bird, R. W. Carrell, F. C. Church, P. B. Coughlin, P. G. Gettins, J. A. Irving, D. A. Lomas, C. J. Luke, R. W. Moyer, et al. 2001. 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