Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin

Teghanemt, Athmane; Re, Fabio; Prohinar, Polonca; Widstrom, Richard; Gioannini, Theresa L.; Weiss, Jerrold

doi:10.1074/jbc.m705994200

Cited by 45 publications

(70 citation statements)

References 41 publications

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“…6A). As previously seen in serum-free mammalian cell culture medium (9,23,24), there was a substantial loss of bioactive sMD-2 during 24 h of incubation in PBS Ϯ 0.1% albumin but no loss of sMD-2 activity during similar incubation in the Express Five TM medium (Fig. 6A).…”

Section: Potent Activation Of Cells Expressing Tlr4 Without Md-2 Requsupporting

confidence: 59%

“…Many factors could contribute to the increased secretion and/or stability of insect cell-derived sMD-2 monomer, including differences in glycosylation (29,30), the lower temperature of culture of insect (27°C) versus mammalian (37°C) cells, and components of the culture medium itself. We have no direct evidence that differences in the metabolic properties of insect versus mammalian cells or the different culture temperatures are important, although time-dependent loss of sMD-2 functional activity at 37°C in serum-free medium has been shown (9,23,24). We also observed time-dependent decay of bioactive sMD-2 derived from insect cells but only when sMD-2 was diluted in PBS Ϯ 0.1% albumin and not when diluted in insect culture medium (Fig.…”

Section: Discussionmentioning

confidence: 57%

“…1A) or in generating monomeric LOS⅐MD-2 (9,24). Comparison of the two conditioned media by SDS-PAGE/immunoblot under nonreducing conditions demonstrated a significant difference in the amount and physical state of sMD-2 in the two media under these conditions.…”

Section: Greater Yield and Stability Of Bioactive Recombinant Human Smentioning

confidence: 99%

“…Time-dependent Decay of Bioactive sMD-2 at 37°C and Protection by Insect Culture Medium-Loss of bioactive sMD-2 has been demonstrated during incubation at 37°C in serum-free medium (9,23,24). In an effort to better understand the apparent protective effects of the insect culture medium on the recovery and preservation of bioactive sMD-2, we compared the stability of insect cell-derived bioactive sMD-2 at 37°C after dilution in either insect culture (Express Five TM ) medium or PBS with or without 0.1% albumin.…”

Section: Potent Activation Of Cells Expressing Tlr4 Without Md-2 Requmentioning

confidence: 99%

“…In contrast to the very stable and potently bioactive properties of the monomeric E⅐MD-2 complex (4, 18), recombinant MD-2 expressed and secreted from mammalian cell cultures, such as human embryonic kidney (HEK) 293 cells, is recovered mainly as inactive multimers with little monomer detected (19 -22). The recovery of active secreted MD-2 (sMD-2) from mammalian cell cultures is further compromised by the instability of sMD-2 at 37°C in serum-free culture medium (9,23,24). In contrast to MD-2 secreted from mammalian cell cultures, nearly 50% of MD-2, produced by insect cells after infection with baculovirus containing MD-2 cDNA, is monomeric * This work was supported, in whole or in part, by National Institutes of Health, United States Public Health Service Grants PO144642 and AI59372 (to J. P. W.).…”

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confidence: 99%

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Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E⅐MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E⅐sCD14 to form monomeric E⅐MD-2 and activate HEK293/ TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E⅐MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E⅐CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.Invasion by Gram-negative bacteria (GNB) 3 is specifically detected and responded to by mammals through mobilization of the innate immune system. In many strains and species of GNB, this process depends on host recognition of and response to the unique complex glycolipid, endotoxin (lipooligosaccharide (LOS) or lipopolysaccharide (LPS)), that comprises much of the outer leaflet of the outer membrane of GNB (1, 2). Minute amounts of endotoxin (E), presented either as an integral part of the outer membrane of GNB or as large aggregates of extracted and purified endotoxin, can stimulate pro-inflammatory responses (2-5). This sensitivity depends upon an ordered series of endotoxin-protein and protein-protein interactions that include the host proteins LPS-binding protein (LBP), membrane-bound or soluble (s)CD14, secreted or TLR4-associated MD-2, and TLR4 (6 -8). Engagement of endotoxin-rich membranes or isolated endotoxin aggregates by LBP facilitates the extraction of endotoxin monomers by CD14 to form monomeric E⅐CD14 complexes that are the most efficient substrate for transfer of endotoxin to MD-2 (4, 6, 9). The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4, 6, 10). MD-2 associates noncovalently with the N-terminal ectodomain of TLR4 and plays a pivotal role not only in TLR4 activation but also in trafficking of TLR4 to the cell surface (7, 11-13). MD-2 also likely interacts transiently with CD14 facilitating transfer of endotoxin monomer from CD14 to MD-2 and, at high molar excess of CD14, reverse transfer of endotoxin from MD-2 to CD14 (14). Simultaneous engagement by MD-2...

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Section: Potent Activation Of Cells Expressing Tlr4 Without Md-2 Requsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 57%

Section: Greater Yield and Stability Of Bioactive Recombinant Human Smentioning

confidence: 99%

Section: Potent Activation Of Cells Expressing Tlr4 Without Md-2 Requmentioning

confidence: 99%

mentioning

confidence: 99%

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Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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Detecting lipopolysaccharide in the cytosol of mammalian cells: Lessons from MD-2/TLR4

Barker

Weiss

2019

Journal of Leukocyte Biology

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Proinflammatory immune responses to Gram-negative bacterial lipopolysaccharides (LPS) are crucial to innate host defenses but can also contribute to pathology. How host cells sensitively detect structural features of LPS was a mystery for years, especially given that a portion of the molecule essential for its potent proinflammatory properties-lipid A-is buried in the bacterial membrane. Studies of responses to extracellular and vacuolar LPS revealed a crucial role for accessory proteins that specifically bind LPS-rich membranes and extract LPS monomers to generate a complex of LPS, MD-2, and TLR4. These insights provided means to understand better both the remarkable host sensitivity to LPS and the means whereby specific LPS structural features are discerned. More recently, the noncanonical inflammasome, consisting of caspases-4/5 in humans and caspase-11 in mice, has been demonstrated to mediate responses to LPS that has reached the host cytosol. Precisely how LPS gains access to cytosolic caspases-and in what form-is not well characterized, and understanding this process will provide crucial insights into how the noncanonical inflammasome is regulated during infection. Herein, we briefly review what is known about LPS detection by cytosolic caspases-4/5/11, focusing on lessons derived from studies of the better-characterized TLR4 system that might direct future mechanistic questions. K E Y W O R D Scaspase, inflammasome, outer membrane vesicle

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The Mammalian Response: A Mosaic of Structures

Williams

2019

Endotoxin Detection and Control in Pharma, Limulus, and Mammalian Systems

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Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin

Cited by 45 publications

References 41 publications

Isolation of Monomeric and Dimeric Secreted MD-2

Isolation of Monomeric and Dimeric Secreted MD-2

Detecting lipopolysaccharide in the cytosol of mammalian cells: Lessons from MD-2/TLR4

The Mammalian Response: A Mosaic of Structures

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