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N-Linked Glycosylation at Asn³ and the Positively Charged Residues within the Amino-Terminal Domain of the C1 Inhibitor Are Required for Interaction of the C1 Inhibitor with Salmonella enterica Serovar Typhimurium Lipopolysaccharide and Lipid A

The CBR Institute for Biomedical Research, Harvard Medical School, 800 Huntington Avenue, Boston, Massachusetts 02115

Received 6 January 2005/ Returned for modification 25 January 2005/ Accepted 2 March 2005

The C1 inhibitor (C1INH), a plasma complement regulatory protein, prevents endotoxin shock, at least partially via the direct interaction of its amino-terminal heavily glycosylated nonserpin region with gram-negative bacterial lipopolysaccharide (LPS). To further characterize the potential LPS-binding site(s) within the amino-terminal domain, mutations were introduced into C1INH at the three N-linked glycosylation sites and at the four positively charged amino acid residues. A mutant in which Asn³ was replaced with Ala was markedly less effective in its binding to LPS, while substitution of Asn⁴⁷ or Asn⁵⁹ had little effect on binding. The mutation of C1INH at all four positively charged amino acid residues (Arg¹⁸, Lys²², Lys³⁰, and Lys⁵⁵) resulted in near-complete failure to interact with LPS. The C1INH mutants that did not bind to LPS also did not suppress LPS binding or LPS-induced up-regulation of tumor necrosis factor alpha mRNA expression in RAW 264.7 macrophages. In addition, the binding of C1INH mutants to diphosphoryl lipid A was decreased in comparison with that of recombinant wild-type C1INH. Therefore, the interaction of C1INH with gram-negative bacterial LPS is dependent both on the N-linked carbohydrate at Asn³ and on the positively charged residues within the amino-terminal domain.

Editor: A. D. O'Brien

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