Biochemistry and Pharmacology - Research Publications
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ItemExploiting information inherent in binding sites of virus-specific antibodies: design of an HCV vaccine candidate cross-reactive with multiple genotypes(INT MEDICAL PRESS LTD, 2006-01-01)BACKGROUND/AIMS: The role of antibody in hepatitis C virus (HCV) infection remains unclear although many reports attest to its role in viral clearance. Here we describe epitopes that are recognized by antibody present in the serum of infected patients and show that such epitopes can induce neutralizing antibodies. METHODS: Human serum containing hyperimmune anti-HCV IgG was used to extract epitopes from a library of synthetic peptides that encompassed the sequences of the E1 and E2 proteins of HCV genotype 1a H77. Peptides that were bound by IgG were identified by mass spectrometry. Assembly of these epitopes with a helper T cell determinant was then carried out in order to construct candidate epitope-based vaccines. RESULTS: Three distinct antigenic sites were defined in the E1E2 glycoproteins by epitopes identified by antibody present in infected individuals. Four of the peptide epitopes identified are conserved in at least three HCV genotypes and are bound by antibody present in the sera of chronically infected and convalescent individuals. Synthetic vaccines based on these epitopes elicited antibodies that are capable of (i) capturing HCV virions from the serum of viraemic patients and (ii) inhibiting HCV pseudovirus particle entry into Huh7 cells. CONCLUSIONS: This approach exploits the information inherent in the binding sites of virus-specific antibodies and represents a novel method for the design of synthetic epitope-based vaccines.
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ItemThymic expression of a gastritogenic epitope results in positive selection of self-reactive pathogenic T cells(AMER ASSOC IMMUNOLOGISTS, 2004-05-15)Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase beta subunit(253-277) (H/Kbeta(253-277)), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kbeta(253-277)) expressed in mice under control of the Ii promoter. The Ii-H/Kbeta(253-277) fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kbeta(253-277) transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kbeta(253-277)-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kbeta(253-277) enhanced positive selection of 1E4-TCR cells in Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kbeta(253-277) mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.
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ItemPromiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells(AMER ASSOC IMMUNOLOGISTS, 2005-11-01)"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.
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ItemCD4(+)CD25(+) regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo(AMER ASSOC IMMUNOLOGISTS, 2006-02-01)A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.
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ItemNon-fibrillar components of amyloid deposits mediate the self-association and tangling of amyloid fibrils(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004-05-14)Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.
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ItemA biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004-11-05)Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.
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ItemEvidence that intracellular beta 1-2 mannan is a virulence factor in Leishmania parasites(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003-10-17)The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.
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ItemIdentification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2006-04-07)All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.