Structural basis of a unique interferon-ß signaling axis mediated via the receptor IFNAR1.

Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-ß (IFN-ß) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-ß can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-ß interaction. The IFNAR1-IFN-ß complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not Ifnar2(-/-) mice, suggesting that IFNAR1-IFN-ß signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-ß.

Modulation of TDM-induced granuloma pathology by human lactoferrin: a persistent effect in mice.

Lactoferrin (LTF), an iron binding protein, is known to exhibit immune modulatory effects on pulmonary pathology during insult-induced models of primary Mycobacterium tuberculosis (Mtb) infection. The effects of LTF correlate with modulation of the immune related development of the pathology, and altering of the histological nature of the physically compact and dense lung granuloma in mice. Specifically, a recombinant human version of LTF limits immediate progression of granulomatous severity following administration of the Mtb cell wall mycolic acid, trehalose 6,6'-dimycolate (TDM), in part through reduced pro-inflammatory responses known to control these events. This current study investigates a limited course of LTF to modulate not only initiation, but also maintenance and resolution of pathology post development of the granulomatous response in mice. Comparison is made to a fusion of LTF with the Fc domain of IgG2 (FcLTF), which is known to extend LTF half-life in circulation. TDM induced granulomas were examined at extended times post insult (day 7 and 14). Both LTF and the novel FcLTF exerted sustained effects on lung granuloma pathology. Reduction of pulmonary pro-inflammatory cytokines TNF-α and IL-1ß occurred, correlating with reduced pathology. Increase in IL-6, known to regulate granuloma maintenance, was also seen with the LTFs. The FcLTF demonstrated greater impact than the recombinant LTF, and was superior in limiting damage to pulmonary tissues while limiting residual inflammatory cytokine production.

Lactoferrin reduces mycobacterial M1-type inflammation induced with trehalose 6,6'-dimycolate and facilitates the entry of fluoroquinolone into granulomas.

Primary infection with Mycobacterium tuberculosis (Mtb) results in the formation of a densely packed granulomatous response that essentially limits the entry and efficacy of immune effector cells. Furthermore, the physical nature of the granuloma does not readily permit the entry of therapeutic agents to sites where organisms reside. The Mtb cell wall mycolic acid, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant molecule for modelling macrophage-mediated events during the establishment of the tuberculosis-induced granuloma pathogenesis. At present, there are no treatments for tuberculosis that focus on modulating the host's immune responses. Previous studies showed that lactoferrin (LF), a natural iron-binding protein proven to modulate inflammation, can ameliorate the cohesiveness of granuloma. This led to a series of studies that further examined the effects of recombinant human LF (rHLF) on the histological progression of TDM-induced pathology. Treatment with rHLF demonstrated significant reduction in size and number of inflammatory foci following injections of TDM, together with reduced levels pulmonary pro-inflammatory cytokines TNF-α and IL-1ß. LF facilitated greater penetration of fluoroquinolone to the sites of pathology. Mice treated with TDM alone demonstrated exclusion of ofloxacin to regions of inflammatory response, whereas the animals treated with rHLF demonstrated increased penetration to inflammatory foci. Finally, recent findings support the hypothesis that this mycobacterial mycolic acid can specifically recruit M1-like polarized macrophages; rHLF treatment was shown to limit the level of this M1-like phenotypic recruitment, corresponding highly with decreased inflammatory response.

Mycobacterial Trehalose 6,6'-Dimycolate-Induced M1-Type Inflammation.

Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b+CD45+ macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis.

Recombinant Human Lactoferrin Reduces Inflammation and Increases Fluoroquinolone Penetration to Primary Granulomas During Mycobacterial Infection of C57Bl/6 Mice.

Infection with Mycobacterium tuberculosis (Mtb) results in the primary formation of a densely packed inflammatory foci that limits entry of therapeutic agents into pulmonary sites where organisms reside. No current therapeutic regimens exist that modulate host immune responses to permit increased drug penetration to regions of pathological damage during tuberculosis disease. Lactoferrin is a natural iron-binding protein previously demonstrated to modulate inflammation and granuloma cohesiveness, while maintaining control of pathogenic burden. Studies were designed to examine recombinant human lactoferrin (rHLF) to modulate histological progression of Mtb-induced pathology in a non-necrotic model using C57Bl/6 mice. The rHLF was oral administered at times corresponding to initiation of primary granulomatous response, or during granuloma maintenance. Treatment with rHLF demonstrated significant reduction in size of primary inflammatory foci following Mtb challenge, and permitted penetration of ofloxacin fluoroquinolone therapeutic to sites of pathological disruption where activated (foamy) macrophages reside. Increased drug penetration was accompanied by retention of endothelial cell integrity. Immunohistochemistry revealed altered patterns of M1-like and M2-like phenotypic cell localization post infectious challenge, with increased presence of M2-like markers found evenly distributed throughout regions of pulmonary inflammatory foci in rHLF-treated mice.

Applications of isotopes in advancing structural and functional heparanomics.

Heparanomics is the study of all the biologically active oligosaccharide domain structures in the entire heparanome and the nature of the interactions among these domains and their protein ligands. Structural elucidation of heparan sulfate and heparin oligosaccharides is a major obstacle in advancing structure-function relationships and heparanomics. There are several factors that exacerbate the challenges involved in the structural elucidation of heparin and heparan sulfate; therefore, there is great interest in developing novel strategies and analytical tools to overcome the barriers in decoding the enigmatic heparanome. This review focuses on the applications of isotopes, both radioisotopes and stable isotopes, in the structural elucidation of the complex heparanome at the disaccharide or oligosaccharide level using liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. This review also outlines the utility of isotopes in determining the substrate specificity of biosynthetic enzymes that eventually dictate the emergence of biologically active oligosaccharides.

Investigating the elusive mechanism of glycosaminoglycan biosynthesis.

Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms.

Using a PyMOL activity to reinforce the connection between genotype and phenotype in an undergraduate genetics laboratory.

With the purpose of developing an activity that would help clarify genetic concepts related to the connection between genotype and phenotype and the nature of mutations, we designed a three hour teaching module using the PyMol software. The activity starts with two pre-laboratory assignments, one to learn how to use PyMol and the other to read about a specific protein or protein family. During the laboratory students are given instructions where and how to find additional information on a specific disease and its causal mutations in order to prepare a 10-minute, in-class presentation. Using a post activity, anonymous quiz, we found a statistically significant different grade distribution in students that participated in the PyMol activity relative to a control group. We also found a significant improvement in the student's comprehension when answering questions regarding the nature of mutations and protein structure. This demonstrates the utility of this simulation activity as a vehicle to improve student's understanding of specific key genetic concepts.

Synthesis and assessment of glycosaminoglycan priming activity of cluster-xylosides for potential use as proteoglycan mimetics.

One of the distinct structural features of many proteoglycans (PGs) is the presence of two or more glycosaminoglycan (GAG) side chains covalently linked to a core protein. Previous studies have shown that the synergistic biological activity of multiple GAG chains, as found in the majority of PGs, cannot be accomplished by the sum of the activities of individual GAG chains. To delineate the biological significance of GAG valency, a number of cluster-xylosides carrying two, three, or four xylose residues on the same scaffold were synthesized using click chemistry. Assessment of cluster-xylosides for their GAG chain priming activity in a cellular system revealed that these cluster-xylosides prime multiple GAG chains per scaffold. Multivalent GAG chains, produced by cluster-xylosides, can better mimic PGs as they carry two or more GAG chains attached to a core protein and therefore can be used as molecular probes to examine the biological significance of GAG multivalency in model organisms.

Dimerized glycosaminoglycan chains increase FGF signaling during zebrafish development.

Proteoglycans (PGs) modulate numerous signaling pathways during development through binding of their glycosaminoglycan (GAG) side chains to various signaling molecules, including fibroblast growth factors (FGFs). A majority of PGs possess two or more GAG side chains, suggesting that GAG multivalency is imperative for biological functions in vivo. However, only a few studies have examined the biological significance of GAG multivalency. In this report, we utilized a library of bis- and tris-xylosides that produce two and three GAG chains on the same scaffold, respectively, thus mimicking PGs, to examine the importance of GAG valency and chain type in regulating FGF/FGFR interactions in vivo in zebrafish. A number of bis- and tris-xylosides, but not mono-xylosides, caused an elongation phenotype upon their injection into embryos. In situ hybridization showed that elongated embryos have elevated expression of the FGF target gene mkp3 but unchanged expression of reporters for other pathways, indicating that FGF/FGFR signaling was specifically hyperactivated. In support of this observation, elongation can be reversed by the tyrosine kinase inhibitor SU5402, mRNA for the FGFR antagonist sprouty4, or FGF8 morpholino. Endogenous GAGs seem to be unaffected after xyloside treatment, suggesting that this is a gain-of-function phenotype. Furthermore, expression of a multivalent but not a monovalent GAG containing syndecan-1 proteoglycan recapitulates the elongation phenotype observed with the bivalent xylosides. On the basis of these in vivo findings, we propose a new model for GAG/FGF/FGFR interactions in which dimerized GAG chains can activate FGF-mediated signal transduction pathways.

Characterization of uniformly and atom-specifically (13)C-labeled heparin and heparan sulfate polysaccharide precursors using (13)C NMR spectroscopy and ESI mass spectrometry.

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D (1)H NMR spectroscopy is often complicated by the extensive signal overlap. (13)C NMR signals, on the other hand, overlap less and therefore, (13)C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific (13)C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly (13)C- and (15)N-labeled precursors were also produced and characterized by (13)C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of (13)C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% (13)C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.