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J. Biol. Chem., Vol. 278, Issue 26, 23624-23629, June 27, 2003
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Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin*
From the
Departments of Microbiology and
Immunology and ¶Biochemistry, Wake Forest
University School of Medicine, Winston-Salem, North Carolina 27157
Received for publication, April 3, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Flagellins from Gram-negative bacteria activate inflammatory cells by a toll-like receptor 5 (TLR5)-dependent signaling pathway. We have examined the interaction between flagellin and TLR5 using an in vitro binding assay. Purified recombinant His-tagged flagellin from Salmonella enteritidis bound to TLR5 in detergent lysates from COS-1 cells transiently transfected with a human TLR5 expression plasmid. Flagellins from Salmonella typhimurium and Escherichia coli also bound to TLR5. The specificity of this interaction was demonstrated by its concentration dependence and lack of TLR5 binding to a biologically inactive form of flagellin or to a His-tagged non-flagellar protein. Flagellin bound to the extracellular domain of TLR5 expressed on the surface of COS-1 cells and to a soluble, monomeric form of the extracellular domain (amino acids 1–636). Although a TLR5 extracellular domain containing amino acids 1–407 retained flagellin binding activity, binding was not evident with a TLR5 peptide encoding residues 1–386. Conversely, a peptide containing amino acid residues 386–636 retained flagellin binding. Thus it is likely that amino acids 386–407 is a binding site for flagellin. This sequence contains a putative leucine-rich repeat. These results support the conclusion that flagellin signaling via TLR5 involves a direct interaction between flagellin and a leucine-rich region in TLR5. We also show that the NH2-terminal 358 amino acids of TLR5 play an important role in its signaling activity. Our results provide, for the first time, a molecular basis for the agonist specificity of a TLR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Activation of the innate immune response by bacteria is mediated by a group of molecules that have collectively been termed modulins (1). This family of molecules includes a wide variety of substances including lipopolysaccharide (LPS)1 from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria, several different viral envelope glycoproteins, and flagellins from Gram-negative bacteria. Monocytes, macrophages, neutrophils, and epithelial cells respond to modulins by producing proinflammatory mediators such as tumor necrosis factor
, interleukin-1
, and nitric oxide. Many bacterial modulins signal
via toll-like receptors (TLR), a family of cell surface molecules that possess
a leucine-rich extracellular domain and a cytoplasmic toll/interleukin-1
receptor homology domain
(2).
Although substantial progress has been made in our understanding of the intracellular pathways involved in TLR signaling, we have very little knowledge of the factors that determine the specificity of individual TLRs. For example, the common structural features that allow a diverse group of agonists such as LPS (3–5), heat shock protein 60 (6), respiratory syncytial virus F protein (7), and Escherichia coli P fimbriae (8) to signal via TLR4 are not readily apparent. Although investigators have accepted the notion that TLRs recognize pathogen-associated molecular patterns (9), we have a limited understanding of the structural elements within TLRs that determine their selectivity for specific agonists. The interaction of LPS with CD14 and TLR4 has been the most extensively studied system. LPS binding to CD14 appears to be required for the subsequent interaction of LPS with TLR4 (10) and MD-2, a TLR4-associated protein that may facilitate or enhance the cell surface expression of TLR4 (11). Difference in LPS structure may influence the quality of these interactions (12). Although the region of TLR4 that is involved in the recognition of LPS has not been defined, the structural features of CD14 that are involved in the recognition of LPS have been well characterized (13–16). In a recent study, it was suggested that peptidoglycan from Staphylococcus aureus can bind directly to the extracellular domain of TLR2 (17). However, soluble CD14 was found to enhance this interaction, leaving open the possibility that CD14 or similar proteins might be mediators of the interaction between peptidoglycan and TLR2.
Recent reports from our laboratory and others have demonstrated that flagellins from Gram-negative bacteria activate a range of inflammatory cells via a TLR5-dependent signaling pathway (18–20). As with other TLRs, flagellin signaling via TLR5 involves the activation of the interleukin-1 receptor-associated kinase (20, 21). Although the TLR5 dependence of Gram-negative flagellin signaling has been well documented, the structural requirements for the interaction between flagellin and TLR5 are only partially understood. Structure/function studies with soluble flagellin indicate that the amino and carboxyl constant regions are most critical for TLR5 signaling (22, 23). We have shown that flagellin is active at concentrations in the picomolar to nanomolar range (24), a finding that is consistent with a very high affinity interaction. However, it was not known if flagellin binds directly to TLR5 or initially interacts with a co-receptor that acts in a manner similar to CD14. We recently developed a robust TLR5 expression system using transiently transfected COS-1 cells to elucidate the mechanism for flagellin-induced self-tolerance (20). Because we have found it difficult to express full-length TLR5 or the extracellular domain of TLR5 in bacteria or yeast,2 we have used the COS-1 expression system to determine whether purified flagellin can bind directly to TLR5 and if so, to define the region of TLR5 required for binding.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cells and Reagents—COS-1 cells and RAW 264.7 cells were maintained in Dulbecco' s modified Eagle's medium with 10% fetal bovine serum and gentamicin. Purified, endotoxin-free recombinant His-tagged Salmonella enteritidis, Salmonella typhimurium, and E. coli flagellins were prepared as previously described (24). In the case of S. typhimurium, we prepared the two types of flagellin, FliC and FljB, that are produced by this strain of bacteria. We also prepared a mutant S. enteritidis flagellin 229 that lacks biological activity (24). Recombinant His-tagged AlgB from Pseudomonas aeruginosa (25) was kindly provided by Dr. Daniel Wozniak (Wake Forest University School of Medicine). Anti-FLAG antibody was obtained from Sigma and high affinity anti-HA antibody from Roche Diagnostics.
Plasmids—The p3XFLAG-CMV-14-TLR5 (20) contains a full-length cDNA encoding human TLR5-FLAG. The HA-tagged human TLR5 expression plasmid was prepared by cloning an amplified TLR5 PCR product into the pMH vector (Roche Diagnostics). A cDNA encoding the signal sequence and extracellular domain of TLR5-ED-(1–636) was generated by PCR using the forward primer, 5'-gccattcccaagcttccgccaatgggagaccacctggacctt-3' and the reverse primer, 5'-gctcgatgcggatccctttaagacttcctcttcatc-3'. The resultant product was directionally cloned into p3XFLAG-CMV-14 at the HindIII and BamHI sites. The truncated forms of the extracellular domain of TLR5 were also generated by PCR and were cloned into the p3XFLAG-CMV vector. All products were sequenced to ensure that mutations were not introduced during the PCR. Although a signal sequence is encoded in the first 28 amino acids of TLR5-(26–28), we have referred to each peptide on the basis of the unprocessed polypeptide (e.g. 1–636). Mutations were introduced into the TLR5 sequence using the QuikChange XL site-directed mutagenesis kit (Stratagene). The presence of mutations was confirmed by sequence analysis. The p3XFLAG-myc-CMV-23 expression vector was obtained from Sigma. Proteins expressed with this vector contain an NH2-terminal preprotrypsin signal sequence followed by a 3x FLAG tag and a COOH-terminal Myc tag.
Binding of Flagellin to TLR5 in Cell Lysates—COS-1 cells (2 x 106) were transiently transfected with TLR5 expression plasmids using FuGENE 6 (Roche Diagnostics) and rested for 48 h prior to use. The binding of flagellin to TLR5 in detergent cell lysates was done in the following manner. Cells were lysed in 0.5 ml of immunoprecipitation buffer containing 0.4% Nonidet P-40 and protease inhibitors (20) and then the lysates were centrifuged at 23,000 x g for 3 min in a refrigerated centrifuge to remove insoluble material. The supernatants were incubated on a rotator in the presence or absence of the indicated concentration of His-tagged flagellin or AlgB and 25 µl of Talon metal affinity resin (Clontech) for2hat4 °C. The samples were centrifuged to remove unbound material and then the Talon beads were washed 3 times with immunoprecipitation buffer, pH 7.5, containing 10 mM imidazole. The samples were then boiled for 5 min in gel sample buffer and electrophoresed in 7.5% SDS-polyacrylamide gels. The proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (100 V, 1 h). Western blot analysis was done as described previously (20) using anti-FLAG or HA antibodies.
Flagellin Binding to Cell Surface-associated TLR5—To assess the binding of flagellin to intact cells, the cultures of transfected COS-1 cells were placed on ice in ice-cold medium in the presence or absence of 4.8 x 10–8 M S. enteritidis flagellin for 30 min at 4 °C. Unbound flagellin was removed by repeated washing of the cell monolayers with ice-cold medium. Cell lysates were prepared as described above and incubated with the Talon metal affinity resin without additional flagellin.
Sedimentation Velocity Measurements of TLR5-ED—Samples were characterized by sucrose density gradient sedimentation as described (29). COS-1 cells were transiently transfected with TLR5-ED-(1–636) or TLR5-ED-(1–425) and lysates were prepared. A 200-µl aliquot of each lysate was layered over a 5-ml 5–20% sucrose gradient and centrifuged for 5 h at 50,000 rpm (20 °C). Fractions (0.5 ml) were collected and analyzed for the presence of TLR5 by Western blotting. The positions of samples within the gradient were converted to sedimentation coefficients based on the profiles of samples of known s20,w value (29).
Inducible Nitric-oxide Synthase Promoter Activation in RAW 264.7 Cells—RAW 264.7 cells are TLR5-negative, but respond to flagellin when transiently transfected with a TLR5 expression plasmid (41). To assess the biological activity of truncated forms of TLR5, RAW 264.7 cells were transiently co-transfected with an expression plasmid encoding full-length human or truncated forms spanning either amino acid residues 386–857 or 426–857, a flagellin-responsive inducible nitric-oxide synthase promoter/luciferase reporter plasmid, and pRL-TK/luciferase (to control for transfection efficiency), rested overnight, and then stimulated for 6 h with 10–10 M flagellin. Inducible nitric-oxide synthase promoter-dependent and constitutive Renilla luciferase activities were measured using the Promega dualluciferase assay system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Flagellin Binds in a Specific Manner to TLR5 in Detergent Lysates from COS-1 Cells—Detergent lysates were prepared from COS-1 cells transiently transfected with an expression plasmid encoding human TLR5-HA and incubated in the presence or absence of either 4.8 x 10–8 M His-tagged flagellin or His-tagged AlgB and Talon metal affinity resin (Fig. 1a). TLR5-HA associated with the Talon resin, but only in the presence of flagellin. Incubation in the absence of flagellin or with the negative control AlgB resulted in background levels of TLR5 association. These results are consistent with a specific interaction between flagellin and TLR5. The extent of complex formation was dependent on the concentration of flagellin. Binding was detected with as low as 8.5 x 10–10 M flagellin (Fig. 1b, lane 2) and was maximal at concentrations above 3.4 x 10–9 M (lane 4). These concentrations of flagellin are in the range of those required for biological activity (20, 21, 24). To obtain additional evidence demonstrating the specificity of the flagellin/TLR5 interaction, we tested the ability of flagellins from S. typhimurium and E. coli as well as a S. enteritidits flagellin mutant 229 that is biologically inactive (24). In the case of S. typhimurium, we tested FliC and FljB, the phase variable forms of flagellin produced by this organism. The two proteins possess equal biological activity. To ensure that flagellin was binding to TLR5 and not the HA tag, we used an expression plasmid encoding TLR5-FLAG. As shown in Fig. 1c, the S. typhimurium and E. coli proteins were all able to bind TLR5. However, the 229 mutant exhibited little if any ability to bind TLR5. Thus these results provide additional support for the specificity of flagellin binding to TLR5.
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Flagellin Binds to the Extracellular Domain of TLR5—Having established the ability of flagellin to bind to TLR5 in cell lysates, we next determined if flagellin bound to TLR5 was expressed on the surface of cells. Transfected COS-1 cells were incubated in the presence or absence of 4.8 x 10–8 M flagellin for 30 min on ice and then washed to remove unbound flagellin. Detergent lysates were then prepared and incubated with the Talon metal affinity resin. As shown in Fig. 1d, flagellin bound to TLR5 expressed on the surface of cells, a finding that is consistent with an interaction involving the extracellular domain of TLR5.
Flagellin Binds to a Soluble Form of the Extracellular Domain of TLR5—As a first step in defining a binding site for flagellin, we evaluated the ability of flagellin to bind to a soluble form of the extracellular domain of TLR5. A cDNA encoding the first 636 amino acids of TLR5 was generated and inserted into the p3XFLAG-CMV-14 expression vector. The expressed protein contains a signal sequence, the extracellular domain of TLR5, and a FLAG tag at the COOH terminus. We have termed the expressed protein, TLR5-ED-(1–636). As shown in Fig. 2a, COS-1 cells not only expressed TLR5-ED-(1–636), but secreted the protein as well (lane 4). Because the cell-associated and secreted forms of the protein exhibit the same apparent molecular mass in SDS gels, we assume that the signal peptide of the cell-associated form is fully processed and the protein was in transit through the secretory pathway. Incubation of TLR5-ED-(1–636)-containing lysates with flagellin and the Talon resin resulted in the association of the protein with the resin (Fig. 2a, lane 2). Association was not observed in the absence of flagellin (lane 1) or in the presence of the 229 flagellin mutant (lane 3). These results not only confirm the ability of flagellin to bind to the extracellular domain of TLR5, but demonstrate a relationship between binding to TLR5 and biological activity (24, 30).
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Determination of the Minimal Form of TLR5-ED Required for Flagellin Binding—Having established the ability of TLR5-ED-(1–636) to bind to flagellin, we initiated experiments to determine the region of the TLR5 extracellular domain that was required for binding. TLR5-ED-(1–478) and TLR5-ED-(1–425), like TLR5-ED-(1–636), bound to flagellin (Fig. 2b). TLR5-ED-(1–425) reproducibly exhibited greater binding activity than the full-length extracellular domain. In contrast, TLR5-ED-(1–372), TLR5-ED-(1–319), and TLR5-ED-(1–159) did not bind to flagellin. It is important to emphasize that all of the proteins were expressed at comparable levels (data not shown). To more precisely determine the minimal polypeptide that retains flagellin binding, we generated a series of truncations between residue 425 and 372 and tested them in the flagellin binding assay. TLR5-ED-(1–407) retained binding activity, but TLR5-ED-(1–386) did not possess detectable activity (Fig. 2c). The activity of the TLR5-ED-(1–407) was reduced relative to the TLR5-ED-(1–425) (Fig. 2c), but was similar to the activity of the full-length extracellular domain, TLR5-ED-(1–636) (Fig. 2b). Based on these results, we hypothesized that the region between amino acid residues 386 and 407 is involved in the binding of flagellin, either by serving as a binding site or by facilitating the folding of the polypeptide in the proper form for flagellin binding.
Having established the importance of the region between residues 386 and 407 of TLR-ED in truncations containing the NH2 terminus of the TLR5 extracellular domain, we tested the converse construct from amino acids 386 to the COOH terminus of the extracellular domain (TLR5-ED-(386–636). As shown in Fig. 2d, TLR5-ED-(386–636), like TLR5-ED-(1–407), possesses the ability to bind flagellin. The fact that the same sequence (386–407) confers binding activity to the NH2 and COOH terminus regions of the extracellular domain of TLR5 supports the idea that the 386–407 region functions as a binding site for flagellin rather than conferring the proper conformation for flagellin binding at some other site. This conclusion is strengthened by the observations that residues 408–636 are not required for flagellin binding by the NH2 terminus TLR5-ED and that residues 1–385 are not required for flagellin binding by the COOH terminus TLR5-ED (Fig. 2).
Sedimentation Velocity Centrifugation of TLR5-ED—We have found that full-length TLR5 self-associates (41). We analyzed the sedimentation velocity of TLR5-ED-(1–636) and TLR5-ED-(1–425) to determine whether these TLR5 extracellular domain forms self-associate or associate with other proteins in the COS-1 cell lysates. We prepared COS-1 cell lysates containing either TLR5-ED-(1–636) or TLR5-ED-(1–425) and used rate zonal centrifugation to determine the sedimentation coefficients for each polypeptide by comparison with standards of known s20,w. As shown in Fig. 3, both polypeptides exhibited experimental s20,w values that were consistent with monomeric forms (6.4 S for TLR5-ED-(1–636) and 4.9 S for TLR5-ED-(1–425)). Thus it is unlikely that the difference between the two polypeptides in terms of flagellin binding is because of differences in their state of self-association. In addition, because the s20,w values are consistent with the presence of monomers, these results support the conclusion that flagellin binds directly to monomeric forms of TLR5-ED and not to an associated protein. However, it is possible that TLR5-ED complexes might form, but are unstable under conditions used in these experiments or that a small subset of complexes containing multimers of TLR-ED and possibly one or more other proteins are responsible for the observed binding of flagellin.
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The TLR5-ED Region Encompassing Amino Acids 1–386 Is Required for Signaling—Because the region encompassing amino acids 386–407 is involved in flagellin binding, we considered the possibility that a TLR5 beginning at residue 386 and including transmembrane and cytoplasmic domains might be sufficient for flagellin signaling. Therefore, we prepared cDNAs encoding residues 386–857 and tested its ability to signal in RAW 264.7 cells, a cell line lacking endogenous TLR5, but responds to flagellin when transiently transfected with a TLR5 expression plasmid (41). Inducible nitric-oxide synthase promoter-dependent luciferase expression was used as a measure of flagellin signaling in the transiently transfected RAW 264.7 cells. Even though the truncated TLR5 contains a flagellin binding site, it did not transmit a signal in the RAW 264.7 cells (Table I).
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Because the truncated TLR5 did not signal, we considered the possibility that the lack of activity was because of a diminished ability of the mutant to self-associate. To evaluate this possibility, COS-1 cells were transiently co-transfected with TLR5-HA and TLR5-FLAG or the truncated form of TLR5-FLAG containing amino acid residues 386–857, and the presence of multimers was assessed following immunoprecipitation with anti-FLAG antibody and Western blotting. As shown in Fig. 4, the full-length form of TLR5-FLAG associates with TLR5-HA. The truncated form of TLR5-FLAG did not appear to be markedly different from full-length TLR5 in its ability to associate with full-length TLR5. Combined with the results presented in Fig. 3, we conclude that the self-association of TLR5 requires the COOH terminus including the transmembrane domain, but signaling also requires regions within the first 385 NH2-terminal amino acids.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Using a COS-1 cell expression system for producing various forms of TLR5, we have shown that flagellin binds in a highly specific manner to the extracellular domain of TLR5. The relationship between the specificity of binding and bioactivity is evidenced by the inability of a biologically inactive form of flagellin to bind TLR5. Using truncated forms of the TLR5 extracellular domain, we have mapped a region (residues 386–407) that is critical for flagellin binding. Although it is formally possible that the requirement for this region is because of its role in the folding of the extracellular domain of TLR5, we favor the hypothesis that this region contains the binding site for flagellin. The ability of TLR5-ED-(1–407) and TLR5-ED-(386–636) to bind flagellin represents the strongest evidence for this conclusion (Fig. 2d). The observation that TLR5-ED-(1–425) binds flagellin better than TLR5-ED-(1–636) or -(1–407) indicates that the region encompassing residues 408–425 plays an accessory role in flagellin binding. It is possible that this region promotes the proper folding of the 386–407 domain or alternatively contributes directly to the quality of the overall binding to flagellin.
Because the TLR5-ED exists as a monomer, it is quite likely that flagellin binds directly to TLR5 and does not require a co-receptor. The observed concentration dependence for flagellin binding is consistent with this conclusion. Although the presence of amino acids 386–407 permits binding to flagellin, signaling is clearly dependent on one or more regions between residues 28 and 386. The absence of this region is associated with a loss of signaling ability (Table I). Because the ability of the truncated TLR5 to oligomerize is not dramatically reduced, we hypothesize that the NH2-terminal domain of the protein plays a role in signaling subsequent to the binding of flagellin. Although our results support the conclusion that flagellin has a high affinity for TLR5, additional studies are clearly required to determine the affinity constant for binding. Our efforts to achieve this goal have been hampered by problems associated with conventional whole cell binding assays and large scale production of TLR5 for in vitro binding assays. In preliminary studies, we observed that flagellin non-specifically interacts with lipid bilayers making it very difficult to assess specific binding to cell-associated TLR5. Furthermore, yeast and bacterial protein expression systems have not been productive sources of full-length TLR5 or its extracellular domain. This contrasts with our ability to obtain expression of the intracellular domain of TLR5.2 Nonetheless, we believe that the results presented in this study represent a major step forward in our understanding of the interaction of flagellin with TLR5 as well as the selectivity of TLR5 for flagellin.
Analysis of the 386–407 region of TLR5 revealed that this sequence
matches the consensus sequence for a leucine-rich repeat (LRR)
(Fig. 5)
(31). The known LRR-containing
proteins share two fundamental properties, namely, the presence of repetitive
LRR sequences and involvement in protein-protein interactions
(31). These LRRs consist of a
short -strand and an -helix that are parallel to each other and
separated by a loop. Although TLRs possess LRRs, the specific function(s) of
this motif has not previously been established. On the basis of our findings,
we propose that the role of this LRR in TLR5 is to serve as a specific binding
site for flagellin.
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Our findings regarding the structural features of TLR5 required for flagellin binding may provide important clues as to the nature of the interaction of these two proteins. The results of two recent studies (22, 23) indicate that the conserved amino and carboxyl regions of flagellin are required for biologic activity. This appears to be in conflict with our observation (24) that a peptide containing only these two regions was inactive. However, as noted by Eaves-Pyles et al. (23), such a peptide may not function because it lacks a bridge that facilitates the necessary interaction between the two ends of the flagellin protein. The involvement of the highly conserved termini of flagellin in TLR5 binding is consistent with the notion that a key feature of pathogen-associated molecular patterns is their invariance between microorganisms of a given class (32). The NH2- and COOH-terminal regions of flagellin possess heptad repeats of hydrophobic amino acid residues (33). It is possible that these sequences play a critical role in the interaction of flagellin and TLR5. For example, Marino et al. (34) have suggested that hydrophobic sequences within a LRR in InIB, a protein involved in Listeria monocytogenes invasion of phagocytes, may play an important role in the interaction of this protein with its cellular receptor.
In the monomeric form, the amino- and carboxyl-terminal regions of
flagellin exist in a relatively disordered form
(35,
36). However, upon
polymerization, the termini become -helical and forms an interlocking
pattern of coiled-coil bundles
(37,
38) within the central core of
the flagellum (39). The
flagellin that is constitutively released by Gram-negative bacteria exists as
predominantly dimers and trimers
(40). This is also the case
with the purified, recombinant flagellin used in our studies. Thus it is
likely that the amino and carboxyl termini of biologically active soluble
flagellin have assumed the formation associated with the polymerized form of
the protein.
Although TLR5-ED-(1–636) and -(1–425) exist predominantly as monomers (Fig. 3), full-length TLR5 forms multimers (Fig. 4 and Ref. 41). Because flagellin exists in large part as dimers and trimers (40), it is possible that a single flagellin multimer may bind to more than one TLR5 oligomer. The ability to cross-link TLR5 complexes may provide an additional mechanism for the extraordinary potency of flagellin.
In view of our findings, we hypothesize that specific LRRs in other TLRs function as binding sites for specific ligands. If subsequent studies support this notion, then it should be possible to develop a family of antagonists that block TLR agonists. Such molecules might be of substantial benefit in the prevention of septic shock. In addition, it should be possible to develop small molecules that recognize the LRR binding motif in specific TLRs and trigger TLR signaling. Such agonists would be of great benefit in those instances in which enhanced innate immunity is needed, as for example, in promoting anti-microbial immunity or increasing the efficacy of vaccination.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI38670 and AI51319. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
smizel{at}wfubmc.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; TLR, toll-like
receptor; TLR5-ED, TLR5-extracellular domain; LRR, leucine-rich repeat; HA,
hemagglutinin.
2 J. Sun and S. B. Mizel, unpublished observations.
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ACKNOWLEDGMENTS |
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We thank Pameeka S. Smith for outstanding technical assistance.
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