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J. Biol. Chem., Vol. 283, Issue 6, 3376-3384, February 8, 2008

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The Differential Impact of Disulfide Bonds and N-Linked Glycosylation on the Stability and Function of CD14^*

Jianmin Meng¹, Peggy Parroche, Douglas T. Golenbock, and C. James McKnight²

From the Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118 and the Department of Medicine, Division of Infectious Disease and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 02118

Innate immunity is the first line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like receptor 4 and MD-2 complex recognize lipopolysaccharide present in the bacterial cell wall. This recognition can be enhanced 100-1000-fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement. In the present study, we use site-directed mutagenesis to address the role of disulfide bonds and N-linked glycosylation on CD14. A differential impact is observed for the five disulfide bonds on CD14 folding, with the first two (Cys⁶-Cys¹⁷ and Cys¹⁵-Cys³²) being indispensable, the third and fourth (Cys¹⁶⁸-Cys¹⁹⁸ and Cys²²²-Cys²⁵³) being important, and the last (Cys²⁸⁷-Cys³³³) being dispensable. A functional role is observed for the first disulfide bond because the C6A substitution severely reduces the ability of CD14 to confer lipopolysaccharide responsiveness to U373 cells. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation, resulting in heterogeneity in CD14 molecular weight. Furthermore, glycosylation at Asn¹³² plays a role in CD14 trafficking and upstream and/or downstream ligand interactions. When mapped onto the crystal structure of mouse CD14, the first two disulfide bonds and Asn¹³² are in close proximity to the initial β strands of the leucine rich repeat domain. Thus, disulfide bonds and N-linked glycosylation in the initial β sheets of the inner concave surface of CD14 are crucial for structure and function.

Received for publication, September 11, 2007 , and in revised form, November 30, 2007.

^* This work was supported in part by National Institutes of Health Grant GM54060 (to D. T. 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 Present address: Dept. of Medicine, University of Massachusetts Medical School, Worcester, MA 01605.

² To whom correspondence should be addressed: 700 Albany St., W407, Boston, MA 01605. Tel.: 617-638-4042; Fax: 617-638-4041; E-mail: cjmck{at}bu.edu.

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