Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling

Kim, Jung-In; Lee, Chang Jun; Jin, Mi Sun; Lee, Cherl‐Ho; Paik, Sang‐Gi; Lee, Hayyoung; Lee, Jie-Oh

doi:10.1074/jbc.m414607200

Cited by 236 publications

(266 citation statements)

References 38 publications

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“…As can be seen from the CD spectrum, CD14-EBD contains a mixture of α-helical and β-sheet type structures. This is confirmed by a recent X-ray structure of mouse sCD14 which depicts a α/β fold for the protein [16]. The spectrum of sCD14-EBD changes minimally upon addition of the endotoxin ligand ReLPS, suggesting there are no global conformational changes in the protein upon ligand binding [22].…”

Section: Expression and Isotopic Labeling Of Scd14 In Pichia Pastorissupporting

confidence: 59%

“…We interpret this as resulting from a change in dynamics of flexible regions of CD14 from fast to slow exchange on the NMR chemical shift time scale, signifying occupancy of various conformational substates selected for binding from within a dynamic ensemble of interconverting population. Interestingly, regions previously identified in endotoxin binding map to a highly flexible hydrophilic rim and also the bottom of a large hydrophobic pocket based on the crystal structure of mouse sCD14 [16]. While the hydrophobic pocket is fairly rigid, the residues on the hydrophilic rim exhibit multiple conformations in addition to high average temperature factors.…”

Section: Changes In Nmr Spectra Of Cd14 Upon Endotoxin Binding Providmentioning

confidence: 99%

“…Deletion mutagenesis experiments and epitope mapping studies with blocking antibodies and proteases have previously identified broad regions on CD14 that are crucial for binding of various microbial ligands (residues 39-44, 57-64, 81-100) and which possess overlapping sites of interaction for these ligands [11][12][13][14][15]. More recently, a highresolution X-ray crystal structure for mouse sCD14 by itself has been determined [16], in which a potential endotoxin binding pocket can be seen at the N-terminus, with the binding regions identified previously from the mutagenesis experiments clustered around this pocket. However, apart from these general interpretations, no direct structural evidence for differential binding to specific residues of CD14 is available, which is critical in order to confirm the role of CD14 as a pattern recognition receptor and provide a structural basis for endotoxin recognition and binding by CD14.…”

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confidence: 99%

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Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Albright¹,

Chen²,

Holbrook³

et al. 2008

Biochemical and Biophysical Research Communications

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CD14 functions as a key pattern recognition receptor for a diverse array of gram-negative and grampositive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy the binding of three different endotoxin ligands, Lipopolysaccharide, lipoteichoic acid and a PGN-derived compound, muramyl dipeptide to a 15 N isotopically labeled 152-residue Nterminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site. KeywordsNMR; human CD14 structure; CD14-endotoxin complex; endotoxin; pattern recognition; pichia pastoris expression; isotopic labeling; cell-based assay; lipopolysaccharide; ReLPS; LTA; BODIPY Clinical manifestation of sepsis typically involves a complex interaction between the host immune system and major bacterial cell wall constituents known as endotoxins. The cluster differentiation antigen, CD14, has been shown to be a key high-affinity cellular receptor for these bacterial endotoxins and plays an important role in endotoxin-induced activation of innate immune cells [1]. In humans, CD14 is constitutively expressed on cell surfaces of monocytes/ macrophages as a 55 kDa membrane-bound protein and also exists in a soluble form (sCD14), in serum and bodily fluids in concentrations of 2-6 μg/ml [2,3]. Recognition and binding of different microbial endotoxins to both mCD14 and sCD14 initiates a signaling cascade mediated by Toll-like receptors (TLRs) that promotes the synthesis and secretion of multiple host-derived inflammatory mediators [4], overactivation of which is ultimately responsible for the deleterious effects of sepsis. Understanding mechanism of endotoxin recognition is

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Section: Expression and Isotopic Labeling Of Scd14 In Pichia Pastorissupporting

confidence: 59%

Section: Changes In Nmr Spectra Of Cd14 Upon Endotoxin Binding Providmentioning

confidence: 99%

mentioning

confidence: 99%

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Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Albright¹,

Chen²,

Holbrook³

et al. 2008

Biochemical and Biophysical Research Communications

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“…A recent study by Kim et al [54] proposed that a large hydrophobic pocket found on the N-terminal side of the CD14 structure is responsible for the binding of the lipid portion of LPS. The CD14-independent activation by R-form LPS suggests that it is either capable of binding directly to the extracellular portion of TLR4/MD-2 or that it integrates into the cell membrane and subsequently binds to the receptor complex.…”

Section: Discussionmentioning

confidence: 99%

R‐form LPS, the master key to the activation ofTLR4/MD‐2‐positive cells

et al. 2006

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Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S-and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-a levels in response to injection of R-and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.Supporting information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2006/35593_pdf IntroductionThe interaction of highly conserved microbial constituents with the innate immune system forms the basis of recognition of and reaction against intruding pathogens in mammals. One such constituent is lipopolysaccharide (endotoxin, LPS), a major recognition marker common to gram-negative bacteria [1][2][3], a large group comprising important human pathogens as well as commensals. Thus, the interaction of LPS with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for an antibacterial defense [3,4]. This primarily protective mechanism may become overshadowed by an acute pathophysiological response with the typical clinical symptoms of septic shock that frequently follows the release of inflammatory mediators, such as tumor necrosis factor (TNF)-a during infection [5][6][7].Innate immunity * Both authors contributed equally to this work. [10]. Binding of allergen to its specific IgE on the surface of MC results in an immediate release of preformed pro-inflammatory mediators (e.g., histamine, TNF-a and proteases) from cytoplasmic granules (degranulation) [11] and a later release of de novo synthesized arachidonic acid metabolites and various cytokines like interleukin 6 (IL-6), TNF-a, and chemokines [10,11]. Unlike allergens, LPS induces no degranulation of MC, but like allergens, it stimulates the de novo synthesis and release of cytokines in these cells [9].Activation of cells by LPS is mediated by the Toll-like receptor 4 (TLR4), a member of the highly conserved protein family of TLR, which are specialized in the recognition of microbial components. In mice, defects in TLR4 result in LPS unresponsiveness [12]. For functional interaction with LP...

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“…LPS must rely on co-receptors such as LBP, MD2 and CD14 to activate the transmembrane TLR4 receptor and initiate signaling. LPS is first bound to soluble LBP and then transferred to CD14 (25)(26)(27), which then acts to shuttle LPS to the TLR4-bound MD2. This direct interaction between MD2 and LPS serves to activate TLR4 and stimulate intracellular signaling.…”

Section: Discussionmentioning

confidence: 99%

Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

Mollen

Gribar

Anand

et al. 2008

Journal of Pediatric Surgery

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Background-The early signaling events in the development of necrotizing enterocolitis (NEC) remain undefined. We have recently shown that the endotoxin (lipopolysaccharide, LPS) receptor Toll-like receptor 4 (TLR4) on enterocytes is critical in the pathogenesis of experimental NEC. Given that the membrane receptor CD14 is known to facilitate the activation of TLR4, we now hypothesize that endotoxemia induces an early up-regulation of CD14 in enterocytes, and that this participates in the early intestinal inflammatory response in the development of NEC.

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Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling

Cited by 236 publications

References 38 publications

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

R‐form LPS, the master key to the activation ofTLR4/MD‐2‐positive cells

Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

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