Activation mechanisms of the innate immune system: structure-function studies of interactions between death domains of MyD88 and IRAK proteins

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 27 de Noviembre de 2012

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of β-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.</p

Functional genomics of the Aeromonas salmonicida lipopolysaccharide O-antigen and A-layer from typical and atypical strains

The A. salmonicida A450 LPS O-antigen, encoded by the wb salmo gene cluster, is exported through an ABC-2 trans porter-dependent pathway. It re presents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. F unctional genomic analysis of this wb salmo region is mostly in agreement with the LPS O-antigen structure of acetylated L -rhamnose (Rha), D -glucose (Glc), and 2-amino-2-deoxy- D -mannose (ManN). Between genes of the wb salmo we found the genes responsible for the biosynthe sis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and at ypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wb salmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wb salmo and A-layer, two major virulence factors, and th is could be the reason they ar e the only ones not found as fish pathogens

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of β-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry

Structural characterization of core Region in Erwinia amylovora lipopolysaccharide.

Erwinia amylovora (E. amylovora) is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS) core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core), wabH and wabG (outer-LPS core mutants). The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry