Identification of LPS-Binding Peptide Fragment of MD-2, a Toll-Receptor Accessory Protein

Manček, Mateja; Pristovšek, Primož; Jerala, Roman

doi:10.1006/bbrc.2002.6748

Cited by 64 publications

(51 citation statements)

References 34 publications

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“…Therefore, N-linked carbohydrate within the amino terminal domain appears to be required for the interaction with LPS. The lipid A-binding motifs of other LPS-interacting proteins contain positively charged amino acids that interact with the negatively charged phosphate groups within lipid A (Ferguson et al, 2000;Lamping et al, 1996;Mancek et al, 2002;Schumann et al, 1997;Visintin et al, 2003). We, therefore, in addition to analyzing the requirement for N-linked carbohydrate, tested whether the four basic amino acids within the amino terminal domain of C1 inhibitor (Arg 18 , Lys 22 , Lys 30 , Lys 55 ) might be involved in the interaction with lipid A.…”

Section: The Interaction Of C1 Inhibitor With Gram Negative Bacterialmentioning

confidence: 99%

C1 inhibitor: Biologic activities that are independent of protease inhibition

Davis

Cai

2007

Immunobiology

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C1 inhibitor therapy improves outcome in several animal models of inflammatory disease. These include sepsis and gram negative endotoxin shock, vascular leak syndromes, hyperacute transplant rejection, and ischemia-reperfusion injury. Furthermore, some data suggest a beneficial effect in human inflammatory disease. In many inflammatory conditions, complement system activation plays a role in pathogenesis. The contact system also very likely is involved in mediation of damage in inflammatory disease. Therefore, the beneficial effect of C1 inhibitor has been assumed to result from inhibition of one or both of these systems. Over the past several years, several other potential anti-inflammatory effects of C1 inhibitor have been described. These effects do not appear to require protease inhibition and depend on non-covalent interactions with other proteins, cell surfaces or lipids. In the first, C1 inhibitor binds to a variety of extracellular matrix components including type IV collagen, laminin, entactin and fibrinogen. The biologic role of these reactions is unclear, but they may serve to concentrate C1 inhibitor at extravascular inflammatory sites. The second is a noncovalent interaction with C3b that results in inhibition of formation of the alternative pathway C3 convertase, a function analogous to that of factor H. The third is an interaction with E and P selectins on endothelial cells that is mediated by the Lewis x tetrasaccharides that are expressed on C1 inhibitor. These interactions result in suppression of leukocyte rolling and transmigration. The fourth interaction is the binding of C1 inhibitor to gram negative bacterial endotoxin that results in suppression of endotoxin shock by interference with the interaction of endotoxin with its receptor complex on macrophages. Lastly, C1 inhibitor binds directly to gram negative bacteria, which leads to suppression of the development of sepsis, as demonstrated in the cecal ligation and puncture model. These observations suggest that C1 inhibitor is a multi-faceted anti-inflammatory protein that exerts its effects through a variety of mechanisms including both protease inhibition and several different non-covalent interactions that are unrelated to protease inhibition.

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Section: The Interaction Of C1 Inhibitor With Gram Negative Bacterialmentioning

confidence: 99%

C1 inhibitor: Biologic activities that are independent of protease inhibition

Davis

Cai

2007

Immunobiology

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“…Mancek et al (17) searched LPS-binding motif of MD-2 by comparing MD-2 with other LPS-binding proteins, and found high amphipathicity and high content of positively charged residues at the region from aa 119 to 132 in human MD-2. The importance of this region (aa 119 -132) was underscored by the studies using MD-2 mutants.…”

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confidence: 99%

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Kobayashi

Saitoh

Tanimura

et al. 2006

The Journal of Immunology

146

108

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LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

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“…Finally, MD-2 can directly bind LPS in some experimental settings (13). Interestingly, a region of human MD-2 spanning aa 119 -132 contains several features common to other LPS-binding proteins (20). This region is rich in basic and aromatic residues that were proposed to interact with the negatively charged and hydrophobic pattern of the LPS molecule.…”

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confidence: 99%

“…This region is rich in basic and aromatic residues that were proposed to interact with the negatively charged and hydrophobic pattern of the LPS molecule. A synthetic peptide derived from this sequence was shown to be able to bind LPS and, remarkably, was also shown to possess antimicrobial activity (20).…”

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confidence: 99%

Separate Functional Domains of Human MD-2 Mediate Toll-Like Receptor 4-Binding and Lipopolysaccharide Responsiveness

Strominger

2003

The Journal of Immunology

100

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Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys95 and Cys105, which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.z

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Identification of LPS-Binding Peptide Fragment of MD-2, a Toll-Receptor Accessory Protein

Cited by 64 publications

References 34 publications

C1 inhibitor: Biologic activities that are independent of protease inhibition

C1 inhibitor: Biologic activities that are independent of protease inhibition

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Separate Functional Domains of Human MD-2 Mediate Toll-Like Receptor 4-Binding and Lipopolysaccharide Responsiveness

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