Sialyltransferases: expression and application for chemo-enzymatic syntheses

Glycosylation is a complex yet common form of post-translational protein/lipid modification in the eukaryotic cells. It is processed by glycosyltransferases (GTs), a large group of enzyme, that are involved in the biosynthesis of glycoprotein and glycolipid sugar chains. Moreover, carbohydrates represent major components of the outer surface of mammalian cells and there is now abundant evidence that terminal glycosylation sequences are involved in adhesion, immune response, and neuronal outgrowth events. Sialylated oligosaccharide sequences have long been predicted to be information – containing molecules and critical determinants, e.g. in cell-cell recognition processes, cell-matrix interactions and maintenance of serum glycoproteins in the circulation. Enzymes responsible for the terminal sialylation are sialyltransferases (STs), a subset of the GT family that use CMP-NeuNAc as the activated sugar donor to catalyze the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. The Myelin-Associated Glycoprotein (MAG), expressed in myelin of the central and peripheral nervous system, has been identified as one of the neurite outgrowth-inhibitory proteins, together with Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). Among all MAG physiological ligands, i.e. brain gangliosides, the GQ1bα is the most potent natural ligand identified so far. Moreover, only the sialic acid containing part of the whole GQ1bα molecule was shown to be important for MAG binding. Therefore, we decided to use a chemo-enzymatic approach for syntheses of the GQ1bα mimetics. For that purpose we expressed recombinant eukaryotic rST3Gal III (EC 2.4.99.6) and hST6Gal I (EC 2.4.99.1), and recombinant prokaryotic Campylobacter jejuni α-2,3/2,8 bifunctional sialyltransferase (Cst-II) using different expression systems. The enzymes were purified and biochemical characterized towards several natural and non-natural acceptor substrates, and used for the preparative chemo-enzymatic synthesis of different carbohydrate structures, e.g. [NeuNAcα(2,8)]NeuNAcα(2,3)Galβ(1,3)GlcNAc-β-OLem; NeuNAcα(2,3)Galβ(1,4)GlcNAc-β-OLem; NeuNAcα(2,3)Galβ(1,4)Glc-β-OLem;)

DRIFT and DRUV spectroscopy methods for studying the interaction of metal compounds with native cellulose

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015Cellulose, the most abundant organic polymer on earth, has numerous applications including the application in pharmacy as excipient in different kinds of pharmaceutical formulations where it comes in contact with metal compounds used in therapeutic purposes. Chemically, is composed of hundreds to thousands of β 1→4 linked D glucopyranose units. It is insoluble in water and most organic solvents and its structure cannot be loosened by heat or with solvents without causing irreversible chemical decomposition. However it can be broken down chemically into glucose units by treatment with concentrated acids at high temperatures. Aqueous solutions of several metals are also known to dissolve cellulose by deprotonating and coordinative binding the hydroxyl groups in the C2 and C3 position of the anxydroglucose. In this work, different metal complexes of copper, iron, vanadium and zinc, and native cellulose were used to a better understanding on the interactions established upon adsorption. Those metals are long used as components of dietary supplements, food additives and different kinds of drugs. It is also known from previous research that the metals complexes can react with cellulose, changing its structure or even cause its decomposition under certain conditions. In general, cellulose fibers have very few functional groups that are capable of bonding with metals and interaction between cellulose and metals and their compounds can occur in one of four ways: adsorption on the fibers, forming chemical bonds with the reactive groups of cellulose, incorporation into cellulose matrix, and formation of complexes with dissolved degradation products of cellulose. The objective of this work was to study the interaction of cellulose fibers with these four metals complexes, evaluating the changes induced by the matrix on the metal complex, specially by the identification of different metal adsorbed species, and assessing the changes in the cellulose structure namely in its crystallinity degree, upon the metal complex adsorption. For the analysis two spectroscopic techniques were used: diffuse reflectance infrared spectroscopy (DRIFTS) and diffuse reflectance ultraviolet-visible spectroscopy (DRUVS). One of the effects that the adsorption of the metal can have in cellulose is by altering its chain spatial arrangement changing its crystallinity degree. The cellulose crystallinity of the samples estimated by DRIFTS, using the region between 1300 and 1180 cm-1 and bands at 898 and 1430 cm-1, showed that in the case of vanadium (V) and zinc (II) there has been increase in the degree of crystallinity while for the other metals crystallinity was not influenced by the presence of metal complexes. However, quantification of this change was only possible in the case of vanadium (V) where it was clear that in the acidic environment at pH 3 and 5 and concentrations higher than 1mM / g cellulose, crystallinity increased from approximately 44 to 76% with the increase of vanadium complex concentration from 1 to 10 mM / g cellulose. The influence of the matrix on the metal complex structure, in the case of vanadium (IV) was analyzed by the alterations observed in the infrared bands, in particular in the regions 1000 -1200 cm-1 and 600 – 670 cm-1, showed that there has been a change in crystalline structure of the initial compound from water soluble α to insoluble β form. The shift of the bands at 1200 cm-1 in all three sets of samples (three different pH) indicated entrapment of the SO42- ion into cellulose and shift in the band at approximately 980 cm-1 indicates entrapment of different vanadium species in the cellulose chains but only in the high range of concentrations. The ultraviolet-visible absorption spectra (DRUVS) shown low intensity of d-d transition bands in the region after 500 nm for the samples with pH 3 and non-adjusted pH, which can be related with the oxidation from V(IV) to V(V). However, considering the blue color of these samples, characteristic for V(IV), the low intensity of the d-d bands in diluted samples appear weak only because they are so in comparison with the stronger CT bands at ~240nm. On the other hand, the high intensity of the CT relative to the d-d bands in samples with cellulose compared to the control may be indicative of binding of cellulose groups to the metal. In the set of samples with pH 5 there are indications of the formation of a new complex and also partial oxidation of V(IV) to V(V). For vanadium (V) adsorbed onto cellulose, the analysis of the new appearing bands present at wavenumbers 960, 971 and 965 cm-1; 836, 829 and 829 cm-1 703, 750 and 747 cm-1 for the non-adjusted pH set of samples, pH 3 and pH 5 sets of samples, respectively, revealed deposition of different polymeric vanadium species onto cellulose when vanadium is present in higher concentrations. Since the equilibrium of vanadium species greatly depends on the pH value it was expected that different species deposited onto cellulose will be observed in the sets of samples with different pH values. At low pH values such as pH 3 it is found that decavanadates are dominant species. At pH 5 dominant species seemed to be mixture of tri and decavanadates and at pH 6-7 we can expect to have mixture of tri and tetravanadates deposited on cellulose. These conclusions are also confirmed after analysis of the DRUV spectra which showed that there hasn’t been any reduction from V (V) to V (IV) and that only different polyoxovanadates are formed in the samples. The DRIFT spectra of iron (II) indicated very weak interaction between the metal complex and the matrix, which means a deposition of the complex on the cellulose surface or on the amorphous phase. Analysis of DRUV spectra showed that it is possible that Fe (III) ions were formed in the samples during oxidation of Fe (II) under aerobic conditions. A different behavior was proposed to iron (III), since in the DRIFT spectra of Fe(III) complex samples (at all preparation pH values) strong bands are appearing at 1384 cm-1 but they are probably just the result of the high complex concentration leading to deposition onto the cellulose exterior surface with low interaction. However, the shifted bands at 1360, 830 and 802 cm-1 indicate deposition of different species or more probably an adsorption onto the cellulose chains with stronger interactions. In the case of zinc and copper metal complexes no significant effect is present in any of the spectra. In fact, neither the bands of the metal complex or those of cellulose are affected by the deposition of the metal complex onto cellulose. Moreover, no new bands are observed, pointing to a poor interaction between the complex and the matrix upon deposition. Since zinc has very low absorptivity in the UV/Vis region no additional information were obtained from the analysis of DRUV spectra. However, the absence of expected d-d bands at around 800 nm in the DRUV spectra of Cu (II) samples, confirmed that the deposition of octahedral copper species onto cellulose did not occurred. Quantitative analysis is performed, for all the metals except zinc, using the Kubelka-Munk equation, or reemission function which gives the correlation between the intensity of the diffuse reflected radiation and concentration for solid samples, similarly to the lambert-Beer law for liquid samples. In all metal complex/cellulose pairs studied the increase of F(R) with increase of concentration was evident. In the full range of concentrations only regression models for V(V) set of samples with pH 5 and V(IV) set with pH 3 showed good fit for the data while in all the other cases only regression models in the lower range of the concentrations from 0.20 up to 1.00, 3.00 or 5.00 mM/g of cellulose (depending of the sample set) showed good fit. These results point to the necessity of optimizing the sample preparation, with a very low repeatability, reducing and replacing the steps involved that leads to a huge possibility of loss of analyte

Clin. Dev. Immunol.

The transcription factors of the Rel/NF-kappa B family function as key regulators of innate and adoptive immunity. Tightly and temporally controlled activation of NF-kappa B-signalling pathways ensures prevention of harmful immune cell dysregulation, whereas a loss of control leads to pathological conditions such as severe inflammation, autoimmune disease, and inflammation-associated oncogenesis. Five family members have been identified in mammals: RelA (p65), c-Rel, RelB, and the precursor proteins NF kappa B1 (p105) and NF-kappa B2 (p100), that are processed into p50 and p52, respectively. While RelA-containing dimers are present in most cell types, c-Rel complexes are predominately found in cells of hematopoietic origin. In T-cell lymphocytes, certain genes essential for immune function such as Il2 and Foxp3 are directly regulated by c-Rel. Additionally, c-Rel-dependent IL-12 and IL-23 transcription by macrophages and dendritic cells is crucial for T-cell differentiation and effector functions. Accordingly, c-Rel expression in T cells and antigen-presenting cells (APCs) controls a delicate balance between tolerance and immunity. This review gives a selective overview on recent progress in understanding of diverse roles of c-Rel in regulating adaptive immunity

“Being in Balance”: Self-Management Experiences Among Young Women with Type 1 Diabetes

Women possess characteristics and experiences unique and different from men. Biological processes such as puberty, menstruation, motherhood and menopause may present challenges to self-management for individuals living with type 1 (T1) diabetes mellitus. In this study, descriptive phenomenology was used to uncover the self-management experiences of nine women aged 22- 30 years living with T1 diabetes. Data collection and analysis occurred simultaneously and followed the methodical structure of van Manen (1997). Study findings revealed five themes: 1) elusiveness of control; 2) dualism of technology; 3) forecasting and maintaining routines; 4) dealing with the “ups and downs”; and, 5) interfacing with the health care team. The essence of the experience for participants revolved around trying to achieve a state of “being in balance.” For these young women, self-management encompassed a desire and need to be in balance with their life and blood sugar levels

The Proteasome System in Infection: Impact of β5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits β1, β2 and β5 and the immunosubunits LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing β1 and β2. In contrast, the β5-propeptide (proβ5) has been suggested to promote preferential integration into β1/β2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proβ5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proβ5 does not direct preferential integration into β1/β2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/β5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proβ5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes

Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7

Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis

Proteasome Inhibitor Bortezomib Ameliorates Intestinal Injury in Mice

Background: Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis. Methodology/Principal Findings: Bortezomib treatment was shown to potently inhibit murine DSS-induced colitis. The attenuation of DSS-induced colitis was associated with decreased inflammatory cell infiltration in the colon. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4 + and CD8 + T cells in the colon and mesenteric lymph nodes. Bortezomib treatment significantly diminished interferon (IFN)-c expression in the colon and mesenteric lymph nodes. Furthermore, cytoplasmic IFN-c production by CD4 + and CD8 + T cells in mesenteric lymph nodes was substantially decreased by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting nuclear factor-kB activation during DSS-induced colitis. Conclusions/Significance: Bortezomib treatment is likely to induce T cell death, thereby suppressing DSS-induced colitis by reducing IFN-c production

Learning from the microbes: exploiting the microbiome to enforce T cell immunotherapy

The opportunities genetic engineering has created in the field of adoptive cellular therapy for cancer are accelerating the development of novel treatment strategies using chimeric antigen receptor (CAR) and T cell receptor (TCR) T cells. The great success in the context of hematologic malignancies has made especially CAR T cell therapy a promising approach capable of achieving long-lasting remission. However, the causalities involved in mediating resistance to treatment or relapse are still barely investigated. Research on T cell exhaustion and dysfunction has drawn attention to host-derived factors that define both the immune and tumor microenvironment (TME) crucially influencing efficacy and toxicity of cellular immunotherapy. The microbiome, as one of the most complex host factors, has become a central topic of investigations due to its ability to impact on health and disease. Recent findings support the hypothesis that commensal bacteria and particularly microbiota-derived metabolites educate and modulate host immunity and TME, thereby contributing to the response to cancer immunotherapy. Hence, the composition of microbial strains as well as their soluble messengers are considered to have predictive value regarding CAR T cell efficacy and toxicity. The diversity of mechanisms underlying both beneficial and detrimental effects of microbiota comprise various epigenetic, metabolic and signaling-related pathways that have the potential to be exploited for the improvement of CAR T cell function. In this review, we will discuss the recent findings in the field of microbiome-cancer interaction, especially with respect to new trajectories that commensal factors can offer to advance cellular immunotherapy