Calprotectin S100A9 Calcium-binding Loops I and II AreEssential for Keratinocyte Resistance to Bacterial Invasion*
Received for publication, August 26, 2008, and in revised form, December 22, 2008 Published, JBC Papers in Press, January 3, 2009, DOI 10.1074/jbc.M806605200
Chantrakorn Champaiboon, Kaia J. Sappington, Brian D. Guenther, Karen F. Ross, and Mark C. Herzberg1
From the Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota,Minneapolis, Minnesota 55455
Epithelial cells expressing calprotectin, a heterodimer ofS100A8 and S100A9 proteins, are more resistant to bacterialinvasion. To determine structural motifs that affect resistanceto bacterial invasion, mutations were constructed in S100A9targeting the calcium-binding loops I and II (E36Q, E78Q,E36Q,E78Q) and theC terminus (S100A91–99 andS100A91–112),which contains putative antimicrobial zinc-binding and phos-phorylation sites. The S100A8 and mutated S100A9 encodingplasmids were transfected into calprotectin-negative KB carci-noma cells. All transfected cells (except KB-sham) expressed27E10-reactive heterodimers. In bacterial invasion assays withListeria monocytogenes and Salmonella enterica serovar Typhi-murium (Salmonella typhimurium), cell lines expressing S100A8in complex with S100A9E36Q, S100A9E78Q, S100A91–99, orS100A91–112mutants or theS100A91–114 (full-length) calprotectinresisted bacterial invasion better than KB-sham.When comparedwith KB-S100A8/A91–114, cells expressing truncated S100A91–99or S100A91–112 with S100A8 also showed increased resistance tobacterial invasion. In contrast, glutamic acid residues 36 and 78 incalcium-binding loops I and II promote resistance in epithelialcells, because cells expressing S100A9E36Q,E78Q with S100A8 wereunable to resist bacterial invasion. Mutations in S100A9 E36Q,E78Q were predicted to cause loss of the calcium-induced pos-itive face in calprotectin, reducing interactions with microtu-bules and appearing to be crucial for keratinocyte resistance tobacterial invasion.
Mucosal keratinocytes continuously confront endogenousand exogenous invading microorganisms. Consequently thesuperficial keratinocytes of the oral mucosa contain a variety ofindigenous bacteria (1). Yet the keratinocytes appear to resistlarge scale invasion and intracellular infection.Expressed in the cytoplasm of squamous mucosal keratino-
cytes, calprotectin (S100A8 and S100A9, MRP8 and MRP14,calgranulin A and B, L1, cystic fibrosis antigen, and 27E10 anti-gen) is a heterodimeric complex of polypeptides of 10.8 and13.2 kDa, respectively (2–4). These two subunits are membersof the S100 protein family, which are involved in cell cycle pro-
gression, cell differentiation, and cytoskeleton-membrane in-teraction (5–7). Calprotectin is the most abundant proteinfound in the cytoplasm of neutrophils (8, 9) and is also found inmonocytes (10), macrophages (11), and human gingival kerati-nocytes (2). Elevated levels of calprotectin have been observedin body fluids such as plasma, saliva, gingival crevicular fluid,stools, and synovial fluid during infections and inflammatoryconditions (12). Consequently, calprotectin is broadly used as amarker for inflammatory bowel diseases (13), reactive arthritis(14), and Sjogren syndrome (15).Functioning as an antimicrobial protein (complex), calpro-
tectin shows broad spectrumactivities againstmicroorganisms,including Capnocytophaga sputigena (16), Candida albicans(17), Escherichia coli, Staphylococcus aureus, Staphylococcusepidermis (18), andBorrrelia burgdorferi (19). Calprotectin alsoinhibits bacterial invasion of epithelial cells by Listeriamonocy-togenes, S. typhimurium, andPorphyromonas gingivalis (20, 21).By promoting resistance to bacterial invasion, calprotectin-ex-pressing cells, including squamous oral keratinocytes, are likelyto contribute to mucosal innate immunity.We have been studying the structural basis of calprotectin-
mediated, cell-associated antimicrobial resistance. UnlikeS100A8 and other members of the S100 family, S100A9 has aextendedC-terminal region, which has an amino acid sequence(residues 89–108) that is identical to the N-terminal region ofneutrophil immobilizing factor (22, 23) and homologous todomain 5 of highmolecular weight kininogen (24). Domain 5 ofhigh molecular weight kininogen has antimicrobial activityagainst E. coli, Pseudomonas aeruginosa, and Enterococcus fae-calis (25). In addition, S100A9 C-terminal residues 103–105form a polyhistidine motif (HHH), which may be involvedin zinc binding (26, 27). Also suggested to be zinc-bindingdomains, the HXXXH motifs in S100A8 and S100A9 are com-monly found in S100 proteins (4, 27, 28). Because zinc isrequired for bacterial growth, either the polyhistidine orHXXXHmotifs have been suggested to bind and sequester zincfrom microorganisms and inhibit bacterial growth (4, 27–29).In addition to zinc, calprotectin chelates other metal ions,including Mn2�, which inhibits growth of S. aureus in tissueabscesses (30).Independent of direct antimicrobial activity, epithelial resist-
ance to invasion may also reflect the ability of bacteria to bindand internalize. Bacterial binding and internalization could beregulated by calprotectin as an interacting partner with thecytoskeleton, although distinguishing fromantimicrobial activ-ity may not always be clear. For example, S100A8/A9 translo-cates across the plasmamembrane and is released from the cell
* This work was supported, in whole or in part, by National Institutes of HealthGrant R01DE11831 (NIDCR). This work was also supported by a scholarshipfrom the Royal Thai Government. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: 17-164 Moos Tower, 515Delaware St. SE, Minneapolis, MN 55455. Fax: 612-626-2651; E-mail:[email protected].
in a tubulin-dependent manner (31). Release from the cell iscontrolled by the penultimate threonine (Thr-113) residue inthe C terminus of S100A9, a substrate for protein kinase C (31).Although tubulin-dependent interactionsmay bring calprotec-tin in proximity to surface bacteria, these interactions couldregulate cytoskeleton-dependent internalization (32).In epithelial cells, calprotectin exists primarily as a het-
erodimeric complex of S100A8 and S100A9 and the individualsubunits are not readily found (2). S100A9 integrity is critical tothe formation of complexes with S100A8 (33) and the calcium-binding loops within the EF-hands contribute to intermolecu-lar stability (4). The calcium-binding loops of S100 proteins alsomodulate intracellular calcium signaling, which affects cell dif-ferentiation, and cell cycle and cytoskeletal interactions (5).Integrity of the S100A9 calcium-binding loopsmay also be crit-ical to resistance against bacterial invasion.We considered that keratinocyte resistance to invasion
reflected the ability of the cells to bind, internalize, and hostviable invaders within the cell. In this study, we hypothesizedthat specific structural motifs of S100A9 in the calprotectincomplex regulate epithelial cell resistance to bacterial invasion.To test this hypothesis, we designed five different S100A9mutant constructs either in the calcium-binding or C-terminaldomains using in vitro site-directed mutagenesis and deletionmutagenesis, respectively. Each mutated S100A9 was thenexpressed in KB cells with S100A8. As we reported previously(20), calprotectin (S100A8/A9) increased the resistance of epi-thelial cells to bacterial invasion. In the presence of S100A8,truncation of the C-terminal domain of S100A9 made the cellsmore resistant to invasion than with full-length S100A9. Incontrast, mutations of S100A9 calcium-binding loops resultedin complete loss of resistance to bacterial invasion. Therefore,the central core polypeptide domain of S100A9 in the calpro-tectin complex plays a crucial role in epithelial resistance tobacterial invasion.
EXPERIMENTAL PROCEDURES
Cells—Wild-type calprotectin-negative KB cells (AmericanType Culture Collection, ATCC CCL-17) were maintained inmodified Eagle’s media (Mediatech, Herndon, VA) supple-mented with 10% fetal bovine serum (Mediatech) in 5% CO2 at37 °C.TransfectedKBcellsweremaintained inmodified Eagle’smedia supplemented with 10% fetal bovine serum and 700�g/mlG418 sulfate (Mediatech). To test the effect of calprotec-tin expression on viable bacteria, mutants and controls weremaintained in medium without G418 sulfate for 4 days beforethe experiments were performed.Bacteria—L. monocytogenes ATCC 10403S (provided by
Dr. Daniel Portnoy, University of California, Berkley) and S.enterica serovar Typhimurium (S. typhimurium) ATCC 14028(provided by Dr. Roy Curtiss III, Washington University, St.Louis) were grown in brain heart infusion medium (Difco) andon tryptic soy agar (Difco) at 37 °C. Listeria and Salmonellawere harvested from log phase or stationary phase, respectively(absorbance of 0.4–0.6 at 620 nm), and used to infect KB cells.Construction of Calprotectin and S100A9Mutant Expressing
KB Cells—The structure of S100A8, S100A9, and mutantconstructs in selected S100A9 functional domains are shown
in Fig. 1, A and B. To construct S100A8 and S100A9 expres-sion vectors, sequences were amplified using the followingprimers: S100A8, sense 5�-GGGCATCATGTTGACCG-AGC-3� and antisense 5�-GTAACTCAGCTACTCTTTGT-GGCTT-3�; S100A9, sense 5�-CGATGACTTGCAAAATG-TCGCAG-3� and antisense 5�-GCCACTGTGGTCTTAG-GGT-3�. To construct truncated S100A9 mutants (Fig. 1B),the sense primer was identical to S100A9 above, and theantisense primers were as follows: S100A91–112, 5�-TTA-GCCCTCCCCGAGGGCTG-3�, and S100A91–99, 5�-TTA-CTCGTCACCCTCGTGCATCTTC-3�. S100A9 mutant se-quences with point mutations in the calcium-binding loops,E36Q and E78Q (Fig. 1B), were constructed using the follow-ing oligonucleotides: S100A9E36Q, 5�-GCACCCTGAACCA-GGGGCAATTCAAAGAGCTGGTGCG-3�, and 5�-CGC-ACCGCCTTGAATTGCCCCTGGTTCAGGGTG-3�, andS100A9E78Q, 5�-GCAGCTGAGTTCGACAGTTCATCAT-GCTGATGGCG-3� and 5�-CGCCATCAGCATGATAAT-GCTCGAAGCCAGCTGC-3�, with the QuickChange�site-directed mutagenesis kit (Stratagene, Rockville, MD).S100A9E36Q,E78Q was constructed using all the oligonucleo-tides from above. PCR products were cloned and amplifiedusing pPCR-Script� (Stratagene, La Jolla, CA). All mutantswere verified by sequencing. S100A8 and mutated S100A9sequences were then cloned into pIRES (BD Biosciences) andpKN-1 (pIRES-EGFP; BD Biosciences with the BamHI site at1887 bp attenuated) plasmids and co-transfected into KBcells using Superfect (Qiagen, Valencia, CA). Transfectantswere selected by resistance to 700 �g/ml G418 sulfate andsorted for enhanced green fluorescent protein expressionusing a FACSorter (BD Biosciences). Cells co-transfectedwith insertless pIRES and pKN-1 served as a sham-controltransfectant (KB-sham). Plasmids containing S100A8 andunmodified S100A9 were co-transfected into KB cells andserved as a positive calprotectin-expressing control (KB-S100A8/A91–114). Stable transfectants were confirmed byreverse transcription-PCR using PCR primers listed above.Immunofluorescence—Cells were grown on coverslips over-
night, washed with PBS,2 and fixed with 4% paraformaldehydefor 10 min at room temperature. Monolayers were washedthree times and permeabilized with 0.2% Triton X-100 for 2min. After washing, monolayers were then incubated withmurine monoclonal antibody against the calprotectin complex(mAb 27E10, diluted 1:50; Bachem, King of Prussia, PA) for 1 hat room temperature, followed by Alexa Fluor 568-conju-gated goat anti-mouse IgG (diluted 1:500; Molecular Probes,Eugene, OR) for 1 h. Both antibodies were diluted in 3% (w/v)bovine serum albumin (Sigma) in PBS. The monolayers werewashed and mounted with Fluoromount G (Southern Bio-technology, Birmingham, AL). Slides were examined usinga Nikon Eclipse epifluorescence microscope and photo-graphed using a Spot digital camera (Diagnostic InstrumentsInc, Sterling Heights, MI).
2 The abbreviations used are: PBS, phosphate-buffered saline; mAb, mono-clonal antibody; CFU, colony-forming unit; PDB, Protein Data Bank; m.o.i.,multiplicity of infection; ELISA, enzyme-linked immunosorbent assay.
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Sandwich ELISA—Todetect calprotectin complex, cells wereresuspended in Hanks’ balanced salt solution (Invitrogen) andsonicated three times on ice at 50 watts for 15 s each (SonifierCell Disruptor W185, Heat Systems, Ultrasonics Inc., Plain-view,MA). To obtain cell cytosol, sonicates were centrifuged at10,000 � g for 20 min, and supernatants were collected, andtotal protein in each sample was determined by BCA proteinassay kit (Pierce). Cell cytosol (50 �g) was analyzed for calpro-tectin using an ELISA. Briefly, 96-well plates were coated over-night at 4 °C with mAb 27E10 (diluted 1:100; Bachem), washedthree times with PBS, pH 7.2, and 0.1% Tween 20, blocked for1 h at 37 °C with blocking buffer (PBS, 0.1% Tween 20 and 0.5mM CaCl2), and washed three more times. Cell cytosol wasadded, incubated for 1 h at 37 °C, andwashed three times. Bioti-nylated murine monoclonal antibody to S100A9 (S 36.48-bio-tin, diluted 1:200; Bachem) was then added and incubated for1 h at 37 °C. Extravidin-horseradish peroxidase and 2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) were usedfor colorimetric detection, and the absorbancewasmeasured at405 nm.Co-immunoprecipitation, Gel Electrophoresis, Silver Stain-
ing, and Western Blotting—mAb 27E10 was used for immuno-precipitation. To demonstrate co-precipitation of S100A8 andmutant S100A9 proteins, products were analyzed on silver-stained gels andWestern blots. In brief, cells were treated withlysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mMEDTA, pH 8.0, 1 mM EGTA, 1% Triton X-100, 0.5% NonidetP-40 with the proteinase inhibitors, 2 �g/ml phenylmethylsul-fonyl fluoride, 1 �g/ml pepstatin A, 1 �g/ml aprotinin, 2.5�g/ml leupeptin). Lysate protein concentration was deter-mined using the BCA protein assay kit (Pierce).In preparation for immunoprecipitation, cell lysates were
pre-cleared (reduced nonspecific binding) using protein A/Gbeads (50 �l), which were previously equilibrated twice in 450�l of cold lysis buffer and centrifuged at 7500 � g for 30–45 s.Cell lysates (1 mg of protein) were incubated with the equili-brated protein A/G beads at 4 °C for 1 h using constant mixingand then centrifuged at 7500 � g for 10 min. The supernatants(pre-cleared lysates) were collected and incubated with mAb27E10 (5 �g) at 4 °C for 1 h with constant mixing. Next, equil-ibrated protein A/G beads (50 �l) were added into the mixtureand incubated overnight at 4 °C with constant mixing, pelletedat 7500� g for 30–45 s, andwashed five times with lysis buffer.Immunoprecipitated protein associated with the beads wasresuspended in 50 �l of 2� SDS-PAGE buffer (1.2 ml of 0.5 MTris, pH 6.8, 2% SDS, 20% glycerol, 0.5 ml of �-mercaptoetha-nol, and 1.6ml of 1% bromphenol blue) and boiled to dissociatethe immunoprecipitated protein from the beads.Immunoprecipitates (30 �l) were analyzed on 15% SDS-
polyacrylamide gels, which were stained with metachromaticsilver following the manufacturer’s instructions (Bio-Rad). ForWestern blotting, KB cell lysates or the immunoprecipitatedsamples were separated on 15% SDS-PAGE, transferred onto a0.2-�m nitrocellulose membrane (Bio-Rad), using a semi-drytransfer apparatus (Bio-Rad), and blocked overnight with 5%nonfatmilk inTBSTbuffer (0.5MNaCl, 20mMTris, pH7.5, and0.1% Tween 20). The membranes were then incubated withmouse anti-human S100A8 monoclonal antibody (C-10, Santa
Cruz Biotechnology, Santa Cruz, CA), mouse anti-humanS100A9monoclonal antibody (S 36.48, Bachem), or rabbit anti-humanS100A9polyclonal antibody (H-90, SantaCruzBiotech-nology) (each diluted 1:500) for 1 h at room temperature,washed three times with TBST buffer, and then incubated witheither horseradish peroxidase-conjugated goat anti-mouse IgGor goat anti-rabbit IgG (diluted 1:1000; Santa Cruz Biotechnol-ogy), respectively, for 1 h at room temperature. After washing,immunoblots were developed using ECL Western blot detec-tion reagents (Amersham Biosciences). Nonspecific isotypeIgG was used as a negative control.Bacterial InvasionAssay—Bacterial invasionwas determined
by the antibiotic protection assay as we described previously(20). In brief, KB transfectants (1.2 � 105 cells) were seededovernight in 24-well plates. Cells were then incubated with L.monocytogenes or S. typhimurium at a multiplicity of infection(m.o.i.) of 100:1 and 1:1, respectively. After 2 h of incubation,the monolayers were washed with Dulbecco’s PBS (Sigma) andincubated in medium containing 100 �g/ml gentamicin(Sigma) for 1.5 h to eliminate extracellular bacteria. Themono-layers were then washed and incubated with sterile distilledwater for 15 min to release intracellular bacteria. Released bac-teria were diluted, plated with a spiral plater (Spiral Biotech,Bethesda,MD), and incubated overnight at 37 °C, and the num-bers of colony-forming units (CFUs) of internalized bacteriawere enumerated on a New Brunswick C-110 colony counter(New Brunswick, NJ). The invasion assay was performed intriplicate and repeated at least three times.Immunofluorescence Analysis of Intracellular and Extracel-
lular Listeria—Cells (1.2� 105) were seeded on glass coverslipsand grown overnight. As described previously (21), the mono-layers were infectedwith L.monocytogenes for 2 h at anm.o.i. of100:1, washed twice with Dulbecco’s PBS, and fixed with 4%paraformaldehyde. Extracellular Listeria were stained usingrabbit anti-Listeria serum (diluted 1:3000; Biodesign, Kenneb-unk, ME) for 1 h, washed with PBS, and then incubated withAlexa Fluor 568-conjugated goat anti-rabbit IgG (diluted 1:500;Molecular Probes) for another hour.All antibodieswere dilutedin 3% bovine serum albumin in PBS. Cells were then permeabi-lized with 0.2% Triton X-100 for 2 min and then stained forboth intracellular and extracellular Listeria. Permeabilizedmonolayers were washed, incubated with rabbit anti-Listeriaserum for 1 h, washed three times, and then incubated withAlexa Fluor 488-conjugated goat anti-rabbit IgG (1:500;Molec-ular Probes) for 1 h. Nuclei were stained using 4�,6�-diamidino-2-phenylindole (diluted 1:3000; Molecular Probes). To verifyantibody specificity, primary antibodieswere replaced by rabbitserum. To determine nonspecific binding, secondary antibod-ies were added without primary antibody. Cells were observedusing aNikon Eclipse fluorescencemicroscope at�400magni-fication, and images from 20 random fields were captured witha Spot digital camera (Diagnostic Instruments Inc.). In eachfield, total Listeria (Alexa 488) and extracellular Listeria (Alexa568) were counted. The number of intracellular Listeria wasdetermined by subtracting the number of extracellular Listeriafrom the total count.Bacterial Binding Assay—Binding of Listeria to KB cells was
performed as described previously (21). Cells (1.2 � 105) were
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seeded on glass coverslips and grown overnight. Monolayerswere then incubated with L. monocytogenes at anm.o.i. of 100:1for up to 60min at 37 °C, washed, and fixed using 4% paraform-aldehyde for 10min. Adherent Listeriawere labeled for 1 hwithrabbit anti-Listeria serum (diluted 1:3000; Biodesign), washed,and incubated for 1 h with Alexa Fluor 568-conjugated goatanti-rabbit IgG. Separate coverslips were incubated with rabbitserum or secondary antibody as controls. At each time point,images from 10 randommicroscopic fields at �200 magnifica-tion were captured with a Spot digital camera, and adherentbacteria were enumerated by visual counting.Structural Analysis of Calcium-free and Calcium-bound
Calprotectin—Because the structure of calcium-free calprotec-tin has not been determined, we generated the homology mod-eled structure using the program MODELLER (34). This pro-gram was chosen because of its ability to handle the alignmentof a heterodimer target sequence (S100A8 and S100A9) witha homodimer structural template. Calcium-free S100A1,S100A4, and S100B structures (PDB codes 1K2H, 1M31, and2PRU, respectively) were chosen as templates for homologymodeling because these proteins have over 30% sequence iden-tity with both S100A8 and S100A9 (35–37). The length andlevel of sequence identity are important factors in accuratehomology model generation. These three structures were used
as templates both individually and combined into a single com-posite template for homology model generation. There is littleoverall difference in the final models, but the structure used inFig. 8 is based on the composite template to remove the bias ofany individual starting structure. Additionally, the residues inthe homology model generated structure were trimmed tomatch those described in PDB code 1XK4 (S100A8 Met-1 toHis-87, S100A9 Lys-4 to Glu-92) to most closely comparechanges in the molecular surface upon calcium binding. Fur-thermore, the absence of electron density for the C-terminal 22residues in S100A9 in PDB code 1XK4 implies that these resi-dues occupymultiple conformations and that there is no exper-imental support to favor a single prediction in our models forthis tail region. The calcium-bound form of calprotectin hasbeen experimentally determined by Skerra and co-workers,PDB code 1XK4 (4), and this information was used in our anal-ysis. The program Swiss-PdbViewer was used to generate theribbon diagrams,molecular surface, and the calculation of elec-trostatic potential with the same settings throughout Fig. 8 (38).The model structure for calcium-bound S100A8 in complexwith calcium-free S100A9 was created by structural superim-position of the N- and C-terminal helices of the calcium-freecalprotectin (composite model structure) onto calcium-boundcalprotectin (PDB code 1XK4). Both structures were written
FIGURE 1. Structure of S100A8, S100A9, and mutations in selected functional domains. A, amino acid sequences of S100A8 and S100A9. Each subunitcontains two EF-hands with helix-loop-helix motifs linked by a hinge region and flanked by N- and C-terminal domains. Calcium-binding loops are in boxes.Putative zinc-binding domains are highlighted in gray. The phosphorylation site is boldface and underlined. Source, NCBI Entrez protein P05109 (A8) andP06702 (A9) (38). B, full-length S100A9 (S100A91–114) and S100A9 mutant constructs, including C-terminal domain deletions (S100A91–112 and S100A91–99) andamino acid substitutions in the calcium-binding loops (S100A9E36Q, S100A9E78Q, and S100A9E36Q,E78Q).
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out as one file followed by removal of the information for cal-cium-free S100A8 and calcium-bound S100A9.Statistical Analyses—Data are presented as the means � S.E.
Significant differences between control (KB-sham) and S100A9mutants were determined using a two-sample Student’s t test.p � 0.05 was considered to be statistically significant.RESULTSFormation of S100A8andMutant S100A9Heterodimers—As
shown schematically in Fig. 1, KB cells were transfected toexpress calprotectin (S100A8/S100A9; Fig. 2B) and S100A8 inthe presence of S100A9 C-terminal deletion mutants (Fig. 2, CandD) or point mutations in the calcium-binding loops (Fig. 2,E–G). Using complex-specific mAb 27E10, S100A8 in thepresence of all mutant S100A9 variants appeared to form cal-
protectin complexes as suggested by immunofluorescencemicroscopy; KB-sham, the sham-transfected control cells, wasnegative (Fig. 2A).Antigen(s) precipitated by mAb 27E10 were recovered from
cytosol of clones expressing calprotectin or S100A8 co-ex-pressed with mutant S100A9 and detected with biotinylatedS100A9mAb (S 36.48-biotin; Bachem) in sandwich ELISA (Fig.3). Truncated variants of S100A9 co-expressed with S100A8and calprotectin appeared to form similar amounts of calpro-tectin complex in KB cell cytosol and significantly more thanthe sham control (*, p � 0.05; **, p � 0.001; Fig. 3, A and B). Inthe same conditions, cells expressing S100A9 point mutationsin calcium-binding loops did not appear to contain cytosolicheterodimers with S100A8 based on reaction with the anti-
GFE
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FIGURE 2. mAb 27E10 reactivity in KB-S100A8/A9 mutants. Monolayers of KB-sham transfectant (A), KB-S100A8/A91–114 (B), KB-S100A8/A91–112 (C),KB-S100A8/A91–99 (D), KB-S100A8/A9E36Q (E), KB-S100A8/A9E78Q (F), and KB-S100A8/A9E36Q,E78Q (G) were fixed with 4% paraformaldehyde and stained asdescribed under “Experimental Procedures.” Monolayers were washed and then permeabilized with 0.2% Triton X-100 for 2 min. Monolayers were incubatedwith mAb 27E10 for 1 h, followed by Alexa Fluor 568-conjugated goat anti-mouse IgG for 1 h at room temperature, which stains calprotectin red. The inset inA shows enhanced green fluorescent protein expressed in KB-sham cells. The experiments were performed three times with similar results. Scale bar, 5 �m.
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S100A9 mAb (Fig. 3, C–E). To learn whether anti-S100A9immunoreactivitywas lostwhenpointmutated S100A9 formedheterodimers with S100A8, immunoprecipitates were analyzedby SDS-PAGE and Western blotting (Fig. 4). On silver-stainedgels, S100A8 and S100A9 (S100A91–114) proteins were visual-ized at 10.8 and 13.2 kDa, respectively (Fig. 4A). Recombinantproteins with similar molecular weights were detected in im-munoprecipitates of S100A8 co-expressed with S100A91–112,S100A9E36Q, S100A9E78Q, and S100A9E36Q,E78Q; S100A8 withS100A91–99 was not well resolved. After immunoprecipita-tion with mAb 27E10, S100A8/A91–114, S100A8/A91–112,S100A8/A91–99, S100A8/A9E36Q, S100A8/A9E78Q, andS100A8/A9E36Q,E78Q resolved on Western blots in reactionwith anti-S100A8 (Fig. 4B). In contrast, anti-S100A9 antibodiesfailed to detect 27E10 immunoprecipitated S100A9E36Q orS100A9E36Q,E78Q (Fig. 4C) or E78Q (data not shown). To confirmthat the anti-S100A9antibodydidnot reactwithS100A9calcium-binding loopmutants, cell lysates were analyzed directly byWest-ern blotting. Lysates from all clones except the sham controlreacted with anti-S100A8 (Fig. 4D). Anti-S100A9 reacted onlywith S100A9 and the C-terminal deletion mutants of S100A9(S100A91–99 and S100A91–112), and failed to react when thecalcium-binding loops weremutated (Fig. 4E). As expected, thecalprotectin complex-specific mAb 27E10 did not react with
either subunit of calprotectin in Western blots (data notshown). In general, clones producing the most heterodimerswere chosen for further study. Clones with S100A9 calcium-binding loop point mutations were selected based on strongsignals with 27E10 in immunofluorescence staining and anti-S100A8 after 27E10 immunoprecipitation.S100A9C-terminal Deletion Increases Resistance to Bacterial
Invasion—To determine whether the C-terminal domain ofS100A9 is crucial for cellular resistance to bacterial invasion,KB-S100A8/A91–112, KB-S100A8/A91–99, and control cellswere incubated for 2 h with either L. monocytogenes ATCC10403S (Fig. 5A) or S. typhimurium ATCC 14028 (Fig. 5B) atm.o.i. 100:1 or 1:1, respectively. When compared with thesham control, KB-S100A8/A91–114, KB-S100A8/A91–112, andKB-S100A8/A91–99 permitted significantly fewer viable intra-cellular Listeria (4.9 and 10.7% invasion, respectively) and Sal-monella (45.5 and 28% invasion, respectively) (p � 0.01). Thenumbers of internalized Listeria and Salmonella in calprotec-tin-negative cells (KB-sham) ranged from 1 � 106 to 3 � 107CFU/well (1 ml) and from 2 � 104 to 4 � 105 CFU/well (1 ml),respectively. KB-S100A8/A91–99 cells showed the greatestresistance to invasion with viable intracellular Listeria andSalmonella, showing 7- and 5-fold fewer intracellular CFUsthan in KB-sham. Remarkably, KB-S100A8/A91–112 and
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FIGURE 3. Calprotectin production in S100A9 mutants. Calprotectin complex in KB-S100A8/A91–112 (A), KB-S100A8/A91–99 (B), KB-S100A8/A9E36Q (C),KB-S100A8/A9E78Q (D), and KB-S100A8/A9E36Q,E78Q (E) were estimated using a sandwich ELISA as described under “Experimental Procedures.” KB-sham andKB-S100A8/A91–114 cells were used as negative and positive controls. Values are means � S.E. (n � 3; *, p � 0.05; **, p � 0.001). For each mutant, at least 10clones were tested. Each experiment was performed in four replicates. The results are representative clones from each mutant.
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KB-S100A8/A91–99 resisted invasion by Listeria more effec-tively than KB-S100A8/A91–114 (p � 0.002 and p � 0.02,respectively; Fig. 5A). KB-S100A8/A91–112 and KB-S100A8/A91–99 also appeared to resist Salmonella invasion moreeffectively than KB-S100A8/A91–114 (p � 0.03 and not sig-nificant, respectively; Fig. 5B).
To learn if the recovered intracellular CFUs reflected thetotal number of intracellular bacteria,Listeriawas stained usinga double immunofluorescence antibody approach. As ex-pected, KB-sham cells contained more intracellular Listeriathan the other clones. The number of intracellular bacteria inKB-sham cells was normalized to 100% invasion for eachday’s experiment. When compared with KB-sham cells, theKB-S100A8/A91–114 cells contained 50% fewer intracellularListeria; the C-terminal mutants, KB-S100A8/A91–112 andKB-S100A8/A91–99, each contained about 75% fewer intracel-lular Listeria (Fig. 5C).S100A9E36Q,E78Q Mutations Ablate Epithelial Resistance to
Bacterial Invasion—To determine whether S100A9 calcium-binding loops I and II contribute to resistance to bacterial inva-sion conferred by calprotectin heterodimer, we quantifiedviable intracellular Listeria after invasion into KB-S100A8/A9E36Q, KB-S100A8/A9E78Q, and KB-S100A8/A9E36Q,E78Qcells. Consistent with the data above, KB-S100A8/A91–114showed greater resistance to invasion by Listeria (50.3% invasion;p � 0.01; Fig. 6A) and Salmonella (59.1% invasion p � 0.001; Fig.6B) than the KB-sham transfectant control. In identical condi-
tions, calprotectin-negative KB-shamcells contained 1 � 106 and 3 � 107CFU/well (1 ml) internalized Listeriaand Salmonella, respectively. Notmarkedly different fromKB-S100A8/A91–114, KB-S100A8/A9E36Q andKB-S100A8/A9E78Q hosted similarlevels of invasion, showing 4-foldfewer viable intracellular Listeria(Fig. 6A) and 1.5–2-fold fewer Sal-monella (Fig. 6B) than KB-sham. Incontrast, KB-S100A8/A9E36Q,E78Qcells, which have mutations in bothS100A9 calcium-binding loops, failto resist invasion by Listeria (99%invasion; Fig. 6A) and Salmonella(93% invasion; Fig. 6B) relative toKB-sham.KB-S100A8/A91–114, KB-S100A8/
A9E36Q, and KB-S100A8/A9E78Qshowed similar percentages of totalintracellular Listeria (ranges from32.9 to 42%), when compared withKB-sham (Fig. 6C). Cells expressingcalprotectin or single mutations inthe calcium-binding loops ofS100A9 co-expressed with S100A8showed significant resistance toinvasion (p � 0.01). Conversely,KB-S100A8/A9E36Q,E78Q, whichexpressed S100A9 with mutations
in both calcium-binding loops, showed a high level of intracel-lular Listeria, similar to KB-sham (Fig. 6C).We next determinedwhether calprotectin-dependent resist-
ance to invasion could be explained by differences in bacterialbinding to the cells. Monolayers were incubated for 15–60minwith L. monocytogenes at an m.o.i. of 100. Cell-associated Liste-ria in nonpermeabilized cells were stained and counted asbound. For all tested KB cell lines, the numbers of bound bac-teria increased with time. At all time points, significantly fewerListeria bound to KB-S100A8/A91–114 and C-terminal mutants(KB-S100A8/A91–112 and KB-S100A8/A91–99; p � 0.05; Fig.7A) or KB-S100A8/A9E36Q (p � 0.05; Fig. 7B) than KB-sham.Listeria bound in similar numbers to KB-S100A8/A91–114 andC-terminal mutants (Fig. 7A). The number of Listeria bound toKB-sham and either KB-S100A8/A9E78Q or KB-S100A8/A9E36Q,E78Q was similar at all time points (Fig. 7B).Calcium-induced Conformational Changes in Calprotectin—
The predicted changes in calprotectin structure and chargeupon calcium binding are shown in Fig. 8. This orientation waschosen to display the calcium-binding sites for both S100A8and S100A9. The ribbon diagram of calcium-free calprotectin,generated by homologymodeling, is shown in Fig. 8A. Calcium-free S100A8 (Fig. 8A, shown in yellow) and A9 (shown in green)contains four helices with the majority of the heterodimerinterface formed by the interactions between the N- and C-ter-minal helices. The corresponding molecular surface for calci-um-free calprotectin and the overall negative charge potential
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FIGURE 4. Analysis of S100A8 and mutated S100A9 in KB transfectants. To analyze the heterodimericcomplexes, the cell lysates (1 mg of protein) from KB-sham, KB-S100A8/A91–114, KB-S100A8/A91–112,KB-S100A8/A91–199, KB-S100A8/A9E36Q, KB-S100A8/A9E78Q, and KB-S100A8/A9E36Q,E78Q were co-immunopre-cipitated (IP) using mAb 27E10. The immunoprecipitated proteins were then separated by 15% SDS-PAGE andeither silver-stained or electroblotted onto nitrocellulose paper (A) and detected with anti-S100A8 (B) andanti-S100A9 antibodies (C), as described under “Experimental Procedures.” Cell lysates were also directly ana-lyzed for S100A8 (D) and S100A9 (E) by Western blots (WB) as described under “Experimental Procedures.” Actinexpression was used as protein loading control (lower panel in D).
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(represented in red) is shown in Fig. 8B. By rotating this view 90°on the z axis, the S100A9 distal end of calprotectin and theoverall negative charge are displayed in Fig. 8C. The ribbondiagramof calcium-bound calprotectinwas generated based onPDB code 1XK4 and is shown in Fig. 8D. The heterodimerinterface is conserved and consists primarily of the N- andC-terminal helices. Calcium binding, however, has induced theformation of an additional helix in the middle of the primarysequence, and these three helices are rotated relative to theirposition in calcium-free calprotectin. Additionally, the C-ter-minal helix expands by several turns upon calcium binding.Binding calcium changes the overall shape of calprotectin (Fig.8E) from approximating a cube to cylindrical with the creationof cleft (indicated by the yellow arrow in Fig. 8E). Furthermore,the calprotectin surface shows more positive potential (repre-sented in blue) with both the creation of a positive patch onS100A8 (Fig. 8E) and an increase in the positive areas of S100A9
(Fig. 8F). The S100A8 calcium-bound structure combined withthe E36Q,E78Q-mutated calcium-free S100A9 structure isshown as a ribbon diagram in Fig. 8G. The predicted effects ofthese mutations on the molecular surface are shown in Fig. 8H.When calcium is bound to calprotectin, the positively chargedface of S100A8 appears unaffected by the E36Q,E78Q muta-tions in S100A9. However, the mutations in S100A9 result inthe loss of the cleft in calprotectin as well as loss of a positivelycharged face on S100A9 (Fig. 8I).
DISCUSSION
We have previously reported that calprotectin can conferresistance to bacterial growth in the cytoplasm of intact cells(20), reduced bacterial binding to the cells, and significantlydecreased invasion (21). How calprotectin protects and con-fers innate immunity to cells against invading microorga-nisms is not known. In vitro, calprotectin antagonizes the
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FIGURE 5. C-terminal deletion of S100A9 increases resistance to Listeria and Salmonella invasion. KB-sham, KB-S100A8/A91–114, KB-S100A8/A91–112, andKB-S100A8/A91–99 were analyzed for bacterial invasion using an antibiotic protection assay as described under “Experimental Procedures.” Monolayers wereincubated with L. monocytogenes ATCC 10403S (A) or S. typhimurium ATCC 14028 (B) at an m.o.i. of 100:1 and 1:1, respectively, for 2 h. Each experiment wasperformed in triplicate wells. Values are means � S.E. of viable intracellular bacteria, relative to KB-sham (100%) from at least three independent experiments.C, immunofluorescence staining for intracellular and extracellular Listeria in KB-sham, KB-S100A8/A91–114, KB-S100A8/A91–112, and KB-S100A8/A91–99 trans-fectants. Monolayers were incubated with L. monocytogenes for 2 h. The intracellular bacteria were enumerated and reported as means � S.E. relative toKB-sham (100%). The results shown are from three independent experiments (*, p � 0.05; **, p � 0.01; #, p � 0.001).
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growth of various microorganisms (16–18, 39). The mecha-nism of antimicrobial activity in vitro is not well understood,but calprotectin has been suggested to inhibit microbialgrowth by chelating Zn2� using HXXXH motifs commonlyfound in S100 proteins and HHH motif of residues 103–105found in the C-terminal domain of S100A9 (27, 29). In thisstudy, we characterized structural domains of S100A9 inassociation with S100A8 that are necessary to regulate epi-thelial cell resistance against invasion by L. monocytogenesand S. typhimurium.Bacterial invasiondepends onbacterial binding to the plasma
membrane, subsequent cytoskeletal rearrangements to facili-tate internalization, and intracellular survival. After invasion,keratinocytes are likely to harbor a mixture of live and deadintracellular bacteria. The antibiotic protection assay was usedto estimate only viable intracellular bacteria that could be enu-merated on agar, whereas immunofluorescence staining wasused to directly visualize and count the total viable and nonvi-
able bacteria within the keratinocytes. If a mutation abrogatedantibacterial activity, a greater proportion of total intracellularListeria (direct counts) was expected to be viable (estimated asCFUs). Indeed,with some exceptions, theCFUs recovered fromthe S100A9mutants in this study mirrored the amount of visu-alized internal Listeria, suggesting that the ablated domainsconfer resistance to invasion through functions other than anti-bacterial activity (see Fig. 6).When the percentage reduction inviable Listeria exceeded the reduction in total bacteria, resist-ance to invasion may be attributable to intracellular antimicro-bial activity and fewer Listeria entering the KB cells (see Fig. 5).These data confirmed that deletion of theC-terminal domain ofS100A9 when co-expressed with S100A8 increased cellularresistance to bacterial invasion when compared with bothKB-S100A8/A91–114 andKB-sham.Otherwork fromour groupsuggests that intracellular anti-Listeria activity might not beapparent until 5–7 h post-invasion (40). Consequently, theshorter term experiments (2 h post-invasion) we report here
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FIGURE 6. Calcium-binding loops of S100A9 and epithelial cell resistance to Listeria and Salmonella invasion. KB-sham, KB-S100A8/A91–114, KB-S100A8/A9E36Q, KB-S100A8/A9E378Q, and KB-S100A8/A9E36Q,E78Q were analyzed for bacterial invasion using an antibiotic protection assay. Monolayers were incubatedwith L. monocytogenes ATCC 10403S (A) or S. typhimurium ATCC 14028 (B) at an m.o.i. of 100:1 and 1:1, respectively, for 2 h. Each experiment was performed intriplicate wells. Values are means � S.E. of viable intracellular bacteria, relative to KB-sham (100%), from at least three independent experiments. C, immuno-fluorescence staining for intracellular and extracellular Listeria in KB-sham, KB-S100A8/A91–114, KB-S100A8/A9E36Q, KB-S100A8/A9E78Q, and KB-S100A8/A9E36Q,E78Q transfectants. Monolayers were incubated with L. monocytogenes for 2 h. The intracellular bacteria were enumerated and reported as means � S.E.relative to KB-sham (100%) as in the legend of Fig. 5. The results shown are from three independent experiments (**, p � 0.01; #, p � 0.001).
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largely identify the role of calprotectin in regulating the bindingand internalization of Listeria and Salmonella. The life cycles ofListeria and Salmonelladiffer, and the 2-h incubation timeusedto study internalization may not be sufficient to observe thedirect intracellular antibacterial effects of calprotectin. None-theless, when compared with KB-sham, calprotectin-express-ing cells are more resistant to invasion by Listeria (4-foldgreater) than Salmonella (2-fold greater). After invasion, Sal-monella remains in vacuoles, whereas Listeria escapes fromendosomes and resides in the cytoplasm (41) where cytoplas-mic calprotectin would be encountered. Short term intracellu-lar calprotectin-mediated anti-Listeria activity could contrib-ute to the resistance to invasion seen in our assays.We first tested KB cells that had been co-transfected with
truncated C-terminal S100A9 constructs and full-lengthS100A8. The extended C terminus of S100A9 may actuallyenable invasion by Listeria and Salmonella because loss of res-idues 113–114 or 100–114 resulted in significantly fewer inter-nalized Listeria and Salmonella than in calprotectin-expressingcells. We had expected that any perturbation of calprotectinwould increase the susceptibility of the cells to invasion. Thisunexpected finding suggests that the C-terminal residues 113and 114 regulate mechanisms that facilitate internalization.Because internalization also reflects the number of bacteriabound, it is important to note that the S100A9 C-terminalmutants also showed about 3-fold fewer bound Listeria overtime than KB-sham (Fig. 7A). S100A9 residue Thr-113 is theonly phosphorylation site in calprotectin, regulating micro-tubule-dependent translocation to the plasma membrane (33),binding (42, 43) and releasing arachidonic acid (44). Therefore,deletion of phosphorylation and arachidonic acid-binding sites
in S100A9 could disrupt calprotectin localization in the plasmamembrane and dysregulate tubulin cytoskeletal events neededto bind and internalize bacteria.The C terminus also includes a portion of a potential zinc-
binding motif, 103HHH105 with potential antimicrobial activity(29). When co-expressed with S100A8, S100A91–112 andS100A91–99 appear to reduce invasion similarly, and differencesin presumptive intracellular anti-Listeria activity could not bedetected. S100A9 has another potential component of a zinc-bindingmotif, 91HEXXH95, which could also contribute to anti-microbial activity (29). In vitro, these domains alone are notsufficient for antimicrobial activity because synthetic peptidescontaining HEXXH and HHH motifs did not inhibit Candidagrowth (29). Antibacterial activity attributable to the zinc-bind-ing motifs could require the presence of the calprotectin com-plex. Yet S100 family members other than calprotectin showantimicrobial activity, including S100A7, S100A12, andS100A15 (45–47). These S100 protein family members do notpossess an extended C-terminal domain with an HHH motif,nor do they appear to form heterodimers. Pretreatment ofS100A7 with zinc did not impact antibacterial activity, andtruncation of the C terminus of S100A7 to delete the zinc-bind-ing HEXXHmotif slightly reduced antibacterial activity in vitro(48). The central core domain of the S100A7 protein, whichincludes a functional EF-hand motif, showed full antibacterialactivity, suggesting that the zinc-binding site in the C terminusof S100A7may be necessary but not sufficient for antimicrobialactivity. As recently reported, the antimicrobial activity of cal-protectin may also depend on chelation of other metal ionssuch as Mn2� (30), but other metal ion-binding motifs in thecomplex have not yet been determined. Hence, it is possiblethat other antimicrobial mechanisms may have been altered bythe S100A9 mutations we report.Complex formation by S100A8 and S100A9 could be neces-
sary for antimicrobial activity and cellular resistance to inva-sion. Calprotectin complex formed with S100A91–99 co-ex-pressed with full-length S100A8 (Figs. 2–4 and Table 1).Similarly, murine S100A91–101 (49), human S100A91–101 (50)and human S100A91–93 (24) each formed heterodimers withS100A8. Hence, heterodimerization into the calprotectin com-plex is independent of the extended C-terminal domain ofS100A9.Calprotectincomplex formationwithS100A8appears tobe sta-
bilized by S100A9 through the C-terminal half of helix IV (adja-cent to theC-terminal domain) or thehydrophobic amino acids inhelix I (see Fig. 1) (9, 49, 51). Each S100 protein has two EF-hands.The canonical C-terminal EF-hand is formed by helices III and IVwith an intervening calcium-binding loop; the N-terminalEF-hand contains a calcium-binding loop betweenhelices I and II.Like other S100 proteins, calprotectin C-terminal EF-hands havehigher affinity for calcium ions than N-terminal EF-hands (6). Tostudy the role of the calcium-binding loops within the EF-hands,we designed two different pointmutations at amino acid residues,Glu-36 and Glu-78, which coordinate calcium ions (52–54). Wesubstituted glutamic acid with glutamine to reduce the calcium-binding affinity of S100A9 (55, 56).The mAb 27E10 specific epitope (85RLTW88) on S100A9 is
clearly intact in all of our mutants (24), suggesting that this
FIGURE 7. Amino acid substitutions in the first and second calcium-bind-ing loops of S100A9 decrease epithelial resistance to Listeria binding.S100A9 C-terminal deletion mutants (KB-S100A8/A91–112 and KB-S100A8/A91–99) (A) and S100A9 calcium-binding loop mutants (KB-S100A8/A9E36Q,KB-S100A8/A9E78Q, KB-S100A8/A9E36Q,E78Q) (B) were incubated with L. mono-cytogenes ATCC 10403S for up to 1 h. Nonadherent bacteria were washed out,and the monolayers were fixed with 4% paraformaldehyde. KB-sham andKB-S100A8/A91–114 were used as negative and positive controls, respectively.Adherent bacteria were stained with specific antibodies and counted asdescribed under “Experimental Procedures.” Values are means � S.E. fromthree independent experiments. (*, p � 0.05; **, p � 0.01).
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antibody serves as a molecule probe for complex formation.S100A9 with mutated calcium-binding loops complexed withS100A8, because transfected KB cells reacted 27E10 as shown
by immunofluorescence microscopy (Fig. 2) and immunopre-cipitation of cell lysates (Fig. 4,A and B). Although calprotectinwith mutated S100A9 calcium-binding Glu-36 and Glu-78 was
FIGURE 8. A representation of the changes in calprotectin structure and charge resulting from S100A9E36Q,E78Q mutations. The ribbon diagram ofcalcium-free and calcium-bound calprotectin, both wild-type and the mutant, is presented in A, D, and G, and the corresponding calculated charged molecularsurface is shown in B, E, and H. The surface of S100A9 is shown in C, F, and I; the view was obtained by rotating the charged molecular surface of calprotectin90° on the z axis, revealing the S100A9 underside. The location of C-terminal tail of S100A9 has not been resolved by crystallography and has been omitted fromthis model. A–C, model structure of calcium-free calprotectin. D–F, calcium-bound form of calprotectin, based on PDB code 1XK4 (4). G–I, S100A8 calcium-bound structure combined with the calcium-free S100A9 structure based upon the E36Q,E78Q mutations. Color key: S100A8, yellow; S100A9, green; calcium,pink; positively charged surface, blue; negatively charged surface, red; and hydrophobic surface, white.
TABLE 1Verification of calprotectin complex formation in S100A9 mutants
a ELISA for calprotectin using mAb 27E10 as capture antibody is shown.b IF indicates immunofluorescence staining for calprotectin using mAb 27E10.c WB indicates Western blotting using anti-S100A8 and anti-S100A9, respectively.d IP indicates immunoprecipitation using mAb 27E10 and then reacted with anti-S100A8 in Western blot or stained with metachromatic silver.
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not detectable by a sandwich ELISA (Fig. 3, C–E), we showclearly that these mutations rendered S100A9 unable to reactwith anti-S100A9 antibody used for detection of calprotectin inthis assay or S100A9 inWestern blots (Fig. 4, C and E). Indeed,we screened several polyclonal and monoclonal anti-S100A9antibodies, and all failed to detect S100A9 with mutated calci-um-binding loops (Table 1). Nonetheless, anti-S100A8 anti-body can detect S100A8 in the presence of S100A9 calcium-binding mutants (Fig. 3D and Table 1). Anti-S100A8 was notuseful as a detection antibody for ELISA. In ELISA, this anti-bodywas unable to detect calprotectin complex in KB-S100A8/A91–114 cells or in C-terminal mutants (data not shown). Usingthe yeast two-hybrid system, S100A9E78Q also appeared to formheterodimers with S100A8, although losing affinity for calcium(54). Consistentwith data obtained using other approaches, ourcalcium-binding loop mutations in S100A9 co-expressed withS100A8 in KB epithelial cells form heterodimeric complexes.Mutations in both calcium-binding loops of S100A9 in
KB-S100A8/A9E36Q,E78Q were sufficient to ablate epithelialresistance to bacterial invasion. The calcium-binding loops ofS100A9 were critical to the ability of calprotectin to resist bac-terial invasion. Although KB-S100A8/A9E36Q and KB-S100A8/A9E78Q with individual calcium-binding loop mutations sus-tained resistance to invasion conferred by calprotectin,mutation of both calcium-binding loops of S100A9 (KB-S100A8/A9E36Q,E78Q) made the KB cells permissive to invasionby Listeria and Salmonella at levels comparable with the shamtransfectant (Fig. 6). Hence, native calcium-binding motifsfunction together to promote resistance to bacterial invasion inKB cells.Calprotectin also affected bacteria at the surface of the KB
cells in a manner dependent on the fidelity of the calcium-binding loops. The pattern of Listeria binding to the KB cellmembrane generally paralleled the pattern of susceptibility orresistance to invasion conferred by the mutant constructs. Thenumber of bacteria bound toKB-S100A8/A9E36Q,E78Qwas sim-ilar to KB-sham but greater than KB-S100A8/A91–114 andS100A8/A9E36Q (Fig. 7B). Although calprotectin generallyaffects invasion by controlling interactions at the cell surface,S100A8/A9E78Q showed comparatively high binding of Listeria(Fig. 7B) but low invasion (Fig. 6). The calprotectin S100A9calcium-binding loops therefore appear to partially regulateepithelial resistance to bacterial invasion by controlling thenumber of bound bacteria that could ultimately be internalized.Although we have previously shown that cellular binding andintracellular growth of invasive bacteria are distinct functionsof calprotectin (21), it remains to be determined whether thecalcium-binding loops contribute to both functions andwhether the interactions are direct or indirect.The binding of calcium by the S100 proteins induces move-
ment and alterations of secondary structural elements, whichgenerate newmolecular surfaces. This is illustrated by compar-ing Fig. 8, A–C (calcium-free) with D–F (calcium-bound).Changing the length and number of helices as well as theirorientations relative to each other brings about this conversionin functional form. The alteration of form is continued as resi-dues are converted from a random coil into a helix, modifyingthe associations between side chains and establishing a new
molecular surface. One example of the new surface is the cre-ation of a cleft (Fig. 8E, arrow) formed in response to bindingcalcium. The adjustment of side chains to a new conformationalters the electrostatic potential of themolecular surface. In thecase of calprotectin, the binding of calcium is responsible forcreating a more positively charged surface.Upon binding calcium, the molecular surface of S100A9
changes potential fromnegative to positive (Fig. 8,C and F), andthe surface of calprotectin mirrors similar changes (B and E).The positively charged face of S100A8 in calprotectin thatforms when calcium is bound may not be involved in bacterialinvasion because this conformation is maintained uponE36Q,E78Q mutation (Fig. 8H).The positive face of S100A9, however, may be critical to bac-
terial invasion (Fig. 8, C and F). The E36Q,E78Q mutations inS100A9 eliminate calcium binding and appears to lock S100A9in a calcium-free conformation. S100A8 should still bind cal-cium. The alteration of the molecular surface and the electro-static potential provides several mechanistic explanations forhow the calcium-binding domain could be involved in mediat-ing resistance to bacterial invasion. The S100A9 E36Q,E78Qmutant loses interactions with any partner that requires a pos-itively charged S100A9 functional surface. For example, nega-tively charged tubulin complexes with calprotectin and isinvolved in bacterial invasion (40). Because the E36Q,E78Qmutations in S100A9 result in loss of this interaction,3 we spec-ulate that the positively charged surface of S100A9 contributesto interactions with tubulin cytoskeleton. Consistent with ourstructural predictions, calprotectin has been shown to contrib-ute to cytoskeletal re-organization and microtubule polymeri-zation in a calcium-dependent manner (4, 54, 57, 58).Ca2�-, Zn2�-, and Cu2�-binding motifs of S100 proteins
generally regulate functional binding to effector molecules (5,6). Calcium binding by calprotectin would be expected to affectconcentrations of intracellular divalent cations, which can alterphosphorylation of specific molecules in downstream signalingcascades. Calprotectinmay also interactwith bacteria at the cellsurface in a manner mimicking S100A12, which shows Ca2�-dependent chaperone/anti-chaperone-like function (59). Cal-protectin resistance to bacterial invasion therefore can involvecomplex downstream responses to calcium-dependent changesin structural motifs in S100A9 in oral keratinocytes.
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