Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of E. coli

Nishikawa, Hanako; Sawasato, Katsuhiro; Mori, Shoko; Fujikawa, Kohki; Nomura, Kaoru; Shimamoto, Keiko; Nishiyama, Kenichi

doi:10.3389/fmolb.2022.986602

Cited by 6 publications

(3 citation statements)

References 53 publications

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“…MPIase consists of the same carbohydrate unit as ECAPG attached to DAG through a pyrophosphate diester bond (75). This molecule is essential and has been implicated in insertion of proteins into the IM in a Secindependent manner and in Sec-dependent protein translocation (74)(75)(76)(77)(78)(79)(80). Unlike ECAPG, the MPIase sugar subunit is built on DAG-PP with the first step being attachment of GlcNAc-P to DAG-P, a reaction catalyzed by CdsA and YnbB (76).…”

Section: Discussionmentioning

confidence: 99%

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Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Morris¹,

Mitchell²

2022

Preprint

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The gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. InEnterobacterales(e.g.,Escherichia, Salmonella, Klebsiella, Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPGcontributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, inEscherichia coliK-12, the reaction synthesizing ECAPGfrom ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPGand provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor.IMPORTANCEThe outer membrane of gram-negative bacteria is a permeability barrier that prevents the entry of many antibiotics into the cell. However, the pathways responsible for outer membrane biogenesis are potential targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane ofEnterobacteralessuch asEscherichia, Salmonella, Klebsiella, andYersinia. ECAPGconsists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate—ECA is the headgroup of a phospholipid. The details of the reaction forming this molecule from ECA linked to an isoprenoid carrier are unknown. We determined that the lipid donor that provides the phospholipid moiety to ECAPGis phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPGprovides the opportunity for the development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat gram-negative infections.

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Section: Discussionmentioning

confidence: 99%

“…It is interesting to compare the synthesis of ECA PG to another glycolipid with a very similar structure, membrane protein intergrase (MPIase) (reviewed in (74)). MPIase consists of the same carbohydrate unit as ECA PG attached to DAG through a pyrophosphate diester bond (75).…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Morris¹,

Mitchell²

2022

Preprint

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“…Membrane protein integrase (MPIase) is a glycolipid found in the inner membrane of E. coli ( Figure 4C ) ( Fujikawa et al, 2022 ; Nishikawa et al, 2022 ). Although its name is unlikely to indicate a glycolipid, it is derived from the unique activity involved in membrane protein integration ( Nishiyama et al, 2006 ; Nishiyama et al, 2010 ; Nishiyama et al, 2012 ).…”

Section: Enterobacterial Common Antigen (Eca) and Membrane Protein In...mentioning

confidence: 99%

Structures, functions, and syntheses of glycero-glycophospholipids

Osawa,

Fujikawa,

Shimamoto

2024

Front. Chem.

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Biological membranes consist of integral and peripheral protein-associated lipid bilayers. Although constituent lipids vary among cells, membrane lipids are mainly classified as phospholipids, glycolipids, and sterols. Phospholipids are further divided into glycerophospholipids and sphingophospholipids, whereas glycolipids are further classified as glyceroglycolipids and sphingoglycolipids. Both glycerophospholipids and glyceroglycolipids contain diacylglycerol as the common backbone, but their head groups differ. Most glycerolipids have polar head groups containing phosphate esters or sugar moieties. However, trace components termed glycero-glycophospholipids, each possessing both a phosphate ester and a sugar moiety, exist in membranes. Recently, the unique biological activities of glycero-glycophospholipids have attracted considerable attention. In this review, we describe the structure, distribution, function, biosynthesis, and chemical synthetic approaches of representative glycero-glycophospholipids—phosphatidylglucoside (PtdGlc) and enterobacterial common antigen (ECA). In addition, we introduce our recent studies on the rare glycero-glyco“pyrophospho”lipid, membrane protein integrase (MPIase), which is involved in protein translocation across biomembranes.

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Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration

Fujikawa

Han

Osawa

et al. 2023

Chemistry A European J

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MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure‐activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone‐like activity of the phosphorylated glycan were observed. These results verified the translocon‐independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

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Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of E. coli

Cited by 6 publications

References 53 publications

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Structures, functions, and syntheses of glycero-glycophospholipids

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration

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