RESUMO
Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.
Assuntos
Proteínas de Fase Aguda , Alanina/metabolismo , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Biblioteca de Peptídeos , Alanina/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Lipopolissacarídeos/antagonistas & inibidores , Mutagênese , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF(31-52) peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-gamma) and IFN-alpha. Analysis of the effect of LALF(31-52) on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF(31-52) protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-alpha levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.