Browsing by Subject "Lectins, C-Type"
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Item The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins(2006-08-11) Cash, Heather Lynn; Hooper, Lora V.Beginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans.Item Molecular Basis of Peptidoglycan Recognition by a Bactericidal Gut Lectin(2010-05-14) Lehotzky, Rebecca Elizabeth; Hooper, Lora V.The mammalian gut is densely populated by varied microbial species. This relationship is mutually beneficial as long as bacteria remain corralled in the gut lumen. The epithelium is protected by the secretion of antimicrobial proteins by specialized epithelial cells in the intestinal crypts. This molecular arsenal includes the RegIII family. RegIII proteins are novel in that they are C-type lectins that directly kill Gram-positive bacteria and thus play a vital role in antimicrobial protection of the mammalian gut. RegIII proteins bind their bacterial targets via interactions with cell wall peptidoglycan, but lack the canonical sequences that support calcium-dependent carbohydrate binding in other C-type lectins. Given these novel functions and the lack of structural clues, nothing was known about the molecular mechanisms by which RegIII family members recognize and bind to peptidoglycan. Furthermore, the question of how RegIII proteins specifically recognize target microbes in the presence of soluble peptidoglycan shed by bacteria in vivo still remained. In this dissertation, I have used NMR spectroscopy as an unbiased approach to study the molecular basis for peptidoglycan recognition by HIP/PAP, a human RegIII lectin. I have shown that HIP/PAP recognizes the peptidoglycan carbohydrate backbone, showing that ligand recognition by RegIII family members is unique compared to other peptidoglycan recognition proteins. This work also shows that HIP/PAP recognizes peptidoglycan in a calciumindependent manner via a conserved ‘EPN’ motif that is critical for bacterial killing. While EPN sequences govern calcium-dependent carbohydrate recognition in other C-type lectins, the unusual location and calcium-independent functionality of the HIP/PAP EPN motif suggest that this sequence is a versatile functional module that can support both calcium-dependent and calciumindependent carbohydrate binding. Further, these studies show that HIP/PAP binding affinity for carbohydrate ligands depends on carbohydrate chain length, supporting a binding model in which HIP/PAP molecules "bind and jump" along the extended polysaccharide chains of peptidoglycan, reducing dissociation rates and increasing binding affinity. I propose that dynamic recognition of highly multivalent carbohydrate epitopes in native peptidoglycan is an essential mechanism governing high affinity interactions between HIP/PAP and the bacterial cell wall.