Normalizing glycosphingolipids restores function in CD4+ T cells from lupus patients

Patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE) have multiple defects in lymphocyte signaling and function that contribute to disease pathogenesis. Such defects could be attributed to alterations in metabolic processes, including abnormal control of lipid biosynthesis pathways. Here, we reveal that CD4+ T cells from SLE patients displayed an altered profile of lipid raft–associated glycosphingolipids (GSLs) compared with that of healthy controls. In particular, lactosylceramide, globotriaosylceramide (Gb3), and monosialotetrahexosylganglioside (GM1) levels were markedly increased. Elevated GSLs in SLE patients were associated with increased expression of liver X receptor β (LXRβ), a nuclear receptor that controls cellular lipid metabolism and trafficking and influences acquired immune responses. Stimulation of CD4+ T cells isolated from healthy donors with synthetic and endogenous LXR agonists promoted GSL expression, which was blocked by an LXR antagonist. Increased GSL expression in CD4+ T cells was associated with intracellular accumulation and accelerated trafficking of GSL, reminiscent of cells from patients with glycolipid storage diseases. Inhibition of GSL biosynthesis in vitro with a clinically approved inhibitor (N-butyldeoxynojirimycin) normalized GSL metabolism, corrected CD4+ T cell signaling and functional defects, and decreased anti-dsDNA antibody production by autologous B cells in SLE patients. Our data demonstrate that lipid metabolism defects contribute to SLE pathogenesis and suggest that targeting GSL biosynthesis restores T cell function in SLE

Structure of human endo-a-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-a-1,2-mannosidase (MANEA) is the sole endoacting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation

Study of ceramide glucosyltransferase

Ceramide glucosyltransferase (CGT) is a key enzyme in glycosphingolipid (GSL) biosynthesis in eukaryotic cells. Inhibition of enzyme activity by an N-alkylated imino sugar, N-butyl-deoxynojirimycin (NB-DNJ), has been evaluated for the therapeutic treatment of inherited glycosphingolipid lysosomal storage diseases. To develop more selective drugs for potential clinical use, further investigation of possible side effects and the design of a more selective inhibitor is required. One concern for clinical use of NB-DNJ is the potential activation of CGT in vivo. When rats were treated with various concentrations of NB-DNJ for 13 weeks to assess the depletion of glycosphingolipids and up-regulation of CGT activity, the reduction of ganglioside levels was observed following an increase in NB-DNJ dose level up to 180 mg/kg/day. However, CGT activity levels were not significantly affected by NB-DNJ treatment. The lack of CGT up-regulation while reducing GSLs by NB-DNJ would be desirable in the clinic to avoid a rapid accumulation of GSLs if patient treatment was concluded. To aid in design of highly selective inhibitors for CGT, enzyme kinetic studies were performed using recombinant human CGT and five different imino sugars. The recombinant enzyme showed similar enzyme kinetics to a native enzyme from HL-60 cells. All the tested imino sugars showed a mixed-type inhibition for ceramide, and an increase in N-alkyl chain provided an improved uncompetitive inhibition. These data suggest that CGT may have two different sites for binding of imino sugars, and the N-alkyl chain length may affect the preference for binding site. When the protein sequence of CGT was analysed using www server programs to predict protein structure, a Rossman fold was predicted in the nucleotide-binding domain as observed in other nucleotide-sugar glycosyltransferase structures. Also, a significant folding similarity to bacterial glycosyltransferase SpsA was predicted. Based on these observations, a possible inhibitor-binding mechanism is discussed that may aid the design of highly selective inhibitors for CGT.</p

Ligand-affinity chromatographic purification of a-fucosidases and a-glucosidases : tools for the structural characterisation of n-linked oligosaccharides from diverse species.

SIGLEAvailable from British Library Document Supply Centre-DSC:DX190836 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Pathways for glycoprotein degradation in mammalian cells

In mammalian cells, N-glycoproteins comprise the bulk of secretory cargo. N-glycosylation is tightly linked to the mechanisms of co- and post-translational folding and quality control in the endoplasmic reticulum (ER). Aberrant glycoproteins must be cleared from the secretory pathway. Several mechanisms for glycoprotein degradation exist, of which ER-associated degradation (ERAD) is most well-studied. The latter involves retrotranslocation of misfolded glycoproteins into the cytosol, followed by proteasomal destruction of the polypeptide. N-linked oligosaccharides are removed from glycoproteins during degradation to form free oligosaccharides (FOS). FOS analysis has been employed to gain a global, rather than single protein-centred view of the pathways of ERAD and the mechanisms of its regulation. It was established that convergent ERAD pathways, distinct in the site of deglycosylation, as well as sensitivity to a number of modulators, exist in the cell. It was further demonstrated that the pathway leading to the production of ER-localised FOS is linked to retrograde Golgi-to-ER transport and is masked by the activity of Golgi endomannosidase. Investigating the involvement on the latter in ERAD has uncovered an endomannosidase-mediated pre-degradative glycoprotein processing route and showed that endomannosidase can rescue a fraction of glycoproteins from degradation following inhibition of ER glucosidases. Further investigation of endomannosidase activity and specificity in different cell lines showed that the ability of the enzyme to process truncated monoglucosylated oligosaccharides appears to be necessary and sufficient for evolutionary conservation in mammals. Overall, the data presented have demonstrated on the global level the connections between the pathways for glycoprotein degradation and processing in the Golgi apparatus, underlining the complexity of biosynthetic regulation in the secretory pathway.This thesis is not currently available via ORA

Peptides as therapeutics

Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo