Removal of Phosphate from Lipid A as a Strategy to Detoxify Lipopolysaccharide

Abstract

Lipopolysaccharide (LPS) may cause sepsis when it enters the blood circulation. The toxic moiety of LPS is the well-preserved lipid A part. Lipid A contains two phosphate groups attached to diglucosamine, which are crucial for the many biological activities of LPS. In previous studies we found that alkaline phosphatase (AP) was able to dephosphorylate LPS. To test whether LPS-dephosphorylation can be used for intervention during sepsis, we investigated the effects of Salmonella minnesota Re 595 LPS and its dephosphorylated counterpart monophosphoryl lipid A (MPLA) on macrophage activation in vivo and in vitro. Exposure of RAW264.7 cells to LPS induced high levels of tumor necrosis factor (TNFalpha) and nitric oxide (NO), whereas MPLA elicited no response. LPS in vivo induced a significant rise in TNFalpha levels in mice and an enhanced inflammatory cell influx in the lung, whereas MPLA did not. Having shown the relevance of this particular phosphate group of LPS, we subsequently explored the LPS-dephosphorylating ability of AP in different tissues, and the effect of AP administration in mice challenged with LPS. Histochemical data show that AP dephosphorylated native LPS in all tissues examined, whereas MPLA was not dephosphorylated. When mice received AP immediately after the LPS challenge, the survival rate was 100%, over 57% in the control group. We conclude that the enzymatic removal of phosphate groups from LPS by AP represents a crucial detoxification reaction, which may provide a new strategy to treat LPS-induced diseases like sepsis.

Prophylactic Treatment with Alkaline Phosphatase in Cardiac Surgery Induces Endogenous Alkaline Phosphatase Release

“…Alkaline phosphatase dephosphorylates and thereby detoxifies not only endotoxins (lipopolysaccharides) but also extracellular nucleotides (2,3). Alkaline phosphatase converts these nucleotides into non-inflammatory nucleosines (4).…”

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations

“…5 and φET or φCET followed by either (4) organic solvent (1-octanol; Szermer-Olearnik and Boratyński, 2015) treatment (OS), (5) detergent Triton X-100; Marcus and Prusky, 1987;Petsch and Anspach, 2000) treatment (TX), (6) enzymatic inactivation of the endotoxin using alkaline phosphatase (Bentala et al, 2002) (AP) or (7) anionexchange chromatography (CIM DEAE disk column (CIM); Adriaenssens et al, 2012).…”

“…An alternative method for the removal of endotoxins is enzymatic inactivation of endotoxins by the removal of the phosphate group of the lipid A fraction by means of alkaline phosphatase (Bentala et al, 2002). Consequently, the treated endotoxin becomes immunologically inactive and should not be detected in a classical Limulus Amebocyte Lysate (LAL) assay or by recombinant factor C (rFC) assays as used in this study.…”

“…The inactivation of endotoxins through enzymatic degradation by means of alkaline phosphatase was described by Bentala et al (2002). Briefly, alkaline phosphatase (rSAP (1,000 U/ml;7.5 U/reaction of 300 µl), New England Biolabs, New England, MA) together with the CutSmart buffer (New England Biolabs; as described by the manufacturer) was added to the phage sample and incubated for 60 min at 37 °C.…”

Reconciliation of IL-1ß loss with TRIF-biased TLR4 signaling by monophosphate lipid A.

“…However, hydrolysis or alkaline phosphatase treatment of LPS yields a monophosphorylated form of Lipid A, known as "Monophosphoryl Lipid A" (MPLA), a TLR4 agonist that displays as little as 0.1-1% as much toxicity as its parent molecule while retaining immuno-stimulatory properties [19][20][21][22][23][24] (see Figure 3 for structural comparisons of Lipid A chemotypes). During the initial years following its discovery, MPLA was found to increase non-specific immune responses to bacterial infection, improving survival of mice subsequently infected with E. coli or S. epidermis [25].…”