The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14

Abstract

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 (Cys 6-Cys17 and Cys15-Cys32) being indispensable, the third and fourth (Cys168-Cys198 and Cys222-Cys253) being important, and the last (Cys 287-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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.