Abstract
Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.
Highlights
Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system
To study the effect of LPS modifications on processing by Alkaline phosphatase (AP), a highly purified E. coli B LPS feedstock sample was first isolated using an extended protocol that included specific steps to remove phospholipid and nucleic acids contaminants that could contribute to background phosphate release
IAP can dephosphorylate both bacterial derived MAMP/PAMPs as well as proinflammatory endogenous damage-associated molecular patterns (DAMPs) of host cell origin produced in response to LPSmediated TLR4/MD2 inflammation
Summary
TLR4/MD2-directed pro-inflammatory cytokine release [15]. Alkaline phosphatase (AP) has far received the most attention as a potential tool to detoxify LPS and attenuate inflammation through lipid A dephosphorylation (16 –19). The potential LPS chemotype substrate specificity spectrum is unknown, as is the unanswered question of which of the many phosphate groups present on LPS are subject to dephosphorylation by IAP. In Salmonella enterica, the regulators controlling LPS remodeling with PEtN/ Ara4N are activated upon exposure to the mouse intestinal lumen environment [37]. These regulatory systems could enable bacterial survival, and block IAP-mediated LPS detoxification. The apparent inability for IAP to dephosphorylate the critical lipid A backbone phosphates in fully acylated substrates suggests the physiological role of IAP in suppressing LPS-induced inflammation may be more complicated and invoke mechanisms beyond regulating TLR4/MD2 receptor activity
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