Purification and Activity of a Wheat 9-kDa Lipid Transfer Protein Expressed in Escherichia coli as a Fusion with the Maltose Binding Protein

We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP.

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The precursor form of the rat liver non-specific lipid-transfer protein, expressed in Escherichia coli , has lipid transfer activity

FEBS Letters ◽

10.1016/0014-5793(92)80374-p ◽

1992 ◽

Vol 296 (2) ◽

pp. 179-183 ◽

Cited By ~ 8

Author(s):

Bernadette C. Ossendorp ◽

Teunis B.H. Geijtenbeek ◽

Karel W.A. Wirtz

Keyword(s):

Escherichia Coli ◽

Rat Liver ◽

Lipid Transfer Protein ◽

Lipid Transfer ◽

Transfer Protein ◽

Transfer Activity ◽

Specific Lipid

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The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-061516-044924 ◽

2018 ◽

Vol 87 (1) ◽

pp. 809-837 ◽

Cited By ~ 51

Author(s):

Bruno Antonny ◽

Joëlle Bigay ◽

Bruno Mesmin

Keyword(s):

Secretory Pathway ◽

Lipid Transfer Protein ◽

Binding Protein ◽

Asymmetric Distribution ◽

Lipid Transfer ◽

Oxysterol Binding Protein ◽

Transfer Protein ◽

Lipid Exchange ◽

Opposite Process ◽

Phosphatidylinositol 4

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle—burning PI(4)P for the vectorial transfer of another lipid—might be general.

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