Neutrophil gelatinase-associated lipocalin: a protein involved in immune defense against microbial pathogens

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Neutrophil gelatinase-associated lipocalin: a protein involved in immune defense against microbial pathogens

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Title: Neutrophil gelatinase-associated lipocalin: a protein involved in immune defense against microbial pathogens
Author: Goetz, David Henry
Abstract: Human neutrophil gelatinase-associated lipocalin was discovered a decade ago as a protein secreted in response to inflammatory stimuli. NGAL was originally described as a component of a disulphide linked heterodimer with gelatinase-B isolated from specific granules secreted by neutrophils. NGAL expression patterns suggested that it plays a role in many processes including cell-growth, apoptosis, cell-differentiation, neoplasia, and defense against bacterial infection. Sequence analysis suggested that NGAL was a member of the lipocalin structural family. The lipocalins are characterized by an eight-stranded beta-barrel, which forms a cup-shaped-generally lipophilic-binding site referred to as the calyx. NGAL was first proposed to function by binding small bacterial hydrophobic chemotactic peptides. However, biochemical data in support of this hypothesis has been difficult to obtain. The aim of my thesis project has been to use structural and biochemical techniques to determine the function of NGAL. My crystal structures of baculovirus-expressed monomer and disulphide-linked homodimer NGAL suggested that NGAL does not in fact bind hydrophobic chemotactic peptides. Subsequently, I serendipitously discovered that NGAL co-purifies with a bacterial chromophore when expressed heterologously in E. coli XL1-Blue. The remainder of my thesis project has been to characterize this chromophore and study its possible relationship to NGAL's in vivo function. I identified the bacterial chromophore as enterobactin and showed that the ligand tightly associates with the NGAL calyx. Enterobactin is a member of a class of iron-binding small molecules known as siderophores that are secreted by microorganisms for the purpose of scavenging iron. My subsequent crystal structure of the NGAL:enterobactin complex revealed that the binding mode exhibits some unique properties. First, NGAL appears to have a binding site that is not only capable of tightly associating with enterobactin but also other larger siderophores. Second, NGAL appears to primarily recognize the catecholate moieties of enterobactin through unique hybrid cation-pi/ionic interactions. These data suggest that NGAL may be part of a general mechanism of the innate immune system for inhibiting bacterial catecholate-siderophore-mediated iron-scavenging in a mammalian host.
Description: Thesis (Ph. D.)--University of Washington, 2002
URI: http://hdl.handle.net/1773/4991

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