N-linked glycosylation is required for c1 inhibitor-mediated protection from endotoxin shock in mice.
C1 inhibitor (C1INH) prevents endotoxin shock in mice via a direct interaction with lipopolysaccharide (LPS). This interaction requires the heavily glycosylated amino-terminal domain of C1INH. C1INH in which N-linked carbohydrate was removed by using N-glycosidase F was markedly less effective in protecting mice from LPS-induced lethal septic shock. N-deglycosylated C1INH also failed to suppress fluorescein isothiocyanate (FITC)-LPS binding to and LPS-induced tumor necrosis factor alpha mRNA expression by the murine macrophage-like cell line, RAW 264.7, and cells in human whole blood. In an enzyme linked immunosorbent assay, the N-deglycosylated C1INH bound to LPS very poorly. In addition, C1INH was shown to bind to diphosphoryl lipid A (dLPA) but only weakly to monophosphoryl lipid A (mLPA). As with intact LPS, binding of N-deglycosylated C1INH to dLPA and mLPA was diminished in comparison with the native protein. Removal of O-linked carbohydrate had no effect on any of these activities. Neither detoxified LPS, dLPA, nor mLPA had any effect on the rate or extent of C1INH complex formation with C1s or on cleavage of the reactive center loop by trypsin. These data demonstrate that N-linked glycosylation of C1INH is essential to mediate its interaction with the LPA moiety of LPS and to protect mice from endotoxin shock.
Liu
Dongxu
D
CBR Institute for Biomedical Research, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gu
Xiaogang
X
Scafidi
Jennifer
J
Davis
Alvin E
AE
D000818:Animals; D002460:Cell Line; D003174:Complement C1 Inactivator Proteins / Q000008:administration & dosage / Q000378:metabolism*; D004195:Disease Models, Animal; D019446:Endotoxemia / Q000139:chemically induced / Q000401:mortality / Q000517:prevention & control*; D006031:Glycosylation; D006801:Humans; D018836:Inflammation Mediators / Q000008:administration & dosage / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*
D003174:Complement C1 Inactivator Proteins; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase