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Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVA To Enable Structure-Activity Analysis.

ABSTRACT: The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP- N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IVA, the first bioactive intermediate in the Lipid A pathway. Our reconstituted multienzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.

SUBMITTER: Sankaranarayanan K

PROVIDER: S-EPMC7206895 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Tunable Enzymatic Synthesis of the Immunomodulator Lipid IV<sub>A</sub> To Enable Structure-Activity Analysis.

Sankaranarayanan Karthik K Antaris Xirui X XX Palanski Brad A BA El Gamal Abrahim A Kao Camilla M CM Fitch William L WL Fischer Curt R CR Khosla Chaitan C

Journal of the American Chemical Society 20190611 24

The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purify ...[more]

PMID: 31184877

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Project description:The immediate immune response to infection by Gram-negative bacteria depends on the structure of a lipopolysaccharide (LPS, also known as endotoxin), a complex glycolipid constituting the outer leaflet of the bacterial outer membrane. Recognition of picomolar quantities of pathogenic LPS by the germ-line encoded Toll-like Receptor 4 (TLR4) complex triggers the intracellular pro-inflammatory signaling cascade leading to the expression of cytokines, chemokines, prostaglandins and reactive oxygen species which manifest an acute inflammatory response to infection. The "endotoxic principle" of LPS resides in its amphiphilic membrane-bound fragment glycophospholipid lipid A which directly binds to the TLR4·MD-2 receptor complex. The lipid A content of LPS comprises a complex mixture of structural homologs varying in the acylation pattern, the length of the (R)-3-hydroxyacyl- and (R)-3-acyloxyacyl long-chain residues and in the phosphorylation status of the ?(1?6)-linked diglucosamine backbone. The structural heterogeneity of the lipid A isolates obtained from bacterial cultures as well as possible contamination with other pro-inflammatory bacterial components makes it difficult to obtain unambiguous immunobiological data correlating specific structural features of lipid A with its endotoxic activity. Advanced understanding of the therapeutic significance of the TLR4-mediated modulation of the innate immune signaling and the central role of lipid A in the recognition of LPS by the innate immune system has led to a demand for well-defined materials for biological studies. Since effective synthetic chemistry is a prerequisite for the availability of homogeneous structurally distinct lipid A, the development of divergent and reproducible approaches for the synthesis of various types of lipid A has become a subject of considerable importance. This review focuses on recent advances in synthetic methodologies toward LPS substructures comprising lipid A and describes the synthesis and immunobiological properties of representative lipid A variants corresponding to different bacterial species. The main criteria for the choice of orthogonal protecting groups for hydroxyl and amino functions of synthetically assembled ?(1?6)-linked diglucosamine backbone of lipid A which allows for a stepwise introduction of multiple functional groups into the molecule are discussed. Thorough consideration is also given to the synthesis of 1,1'-glycosyl phosphodiesters comprising partial structures of 4-amino-4-deoxy-?-L-arabinose modified Burkholderia lipid A and galactosamine-modified Francisella lipid A. Particular emphasis is put on the stereoselective construction of binary glycosyl phosphodiester fragments connecting the anomeric centers of two aminosugars as well as on the advanced P(III)-phosphorus chemistry behind the assembly of zwitterionic double glycosyl phosphodiesters.

Dataset Information

Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVA To Enable Structure-Activity Analysis.

Publications

Tunable Enzymatic Synthesis of the Immunomodulator Lipid IV<sub>A</sub> To Enable Structure-Activity Analysis.

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