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J. Biol. Chem., Vol. 282, Issue 11, 7877-7884, March 16, 2007

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Endotoxin-binding Proteins Modulate the Susceptibility of Bacterial Endotoxin to Deacylation by Acyloxyacyl Hydrolase^*

Theresa L. Gioannini^¶1, Athmane Teghanemt, DeSheng Zhang, Polonca Prohinar, Erika N. Levis, Robert S. Munford^||, and Jerrold P. Weiss^**

From the Inflammation Program, Departments of Internal Medicine, Biochemistry, and ^**Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, ^¶Veterans Affairs Medical Center, Iowa City, Iowa 52246, and ^||Department of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444–16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579–36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOS_agg or LPS_agg) ± LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14 endotoxin aggregates (E_agg):LBP (molar ratio of E/LBP 100:1) >>E_agg, E_agg:LBP (E/LBP 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.

Received for publication, May 25, 2006 , and in revised form, January 8, 2007.

^* This work was supported by U.S. Public Health Service Grants PO144642 and AI59372 (to J. P. W.) and AI18188 (to R. S. M.) and a Veterans Affairs Merit Review grant (to T. L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Roy J. and Lucille A. Carver College of Medicine, University of Iowa and the Veterans Affairs Medical Center, Inflammation Program, Oakdale Research Campus, 2501 Crosspark Rd., Coralville, IA 52241. E-mail: theresa-gioannini{at}uiowa.edu.

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