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Advanced Glycation Endproducts
Published in Sara C. Zapico, Mechanisms Linking Aging, Diseases and Biological Age Estimation, 2017
Andreas Simm, Alexander Navarrete Santos
Cells possess specific binding and receptor molecules for AGEs (Ott et al. 2014). The major and best known receptor for AGEs is the “Receptor for AGEs”, RAGE or synonymous AGER. But other binding proteins have also been described. These are oligosaccharyltransferase (OST48, AGE-R1), 80K-H (AGE-R2) (Li et al. 1996), galectin-3 (AGE-R3) (Vlassara et al. 1995), CD36 (Kuniyasu et al. 2003, Ohgami et al. 2001, Ohgami et al. 2002), and scavenger receptors IIa and −b (Takata et al. 1988, 1989). RAGE is a member of the immunoglobulin receptor family and binds several ligands such as AGEs, HMGB-1, S100 proteins or amyloid beta peptide. Binding of agonists like the AGEs to RAGE results in activation of NADPH-oxidases and other less well described pathways that lead to increased production of ROS. The major downstream target of RAGE is the proinflammatory NFκB-pathway, which in turn leads to elevated RAGE expression and perpetuation of the cellular inflammatory state (Bierhaus et al. 2006). This inflammatory state is characterized by the production of inflammatory cytokines such as IL-6 and TNFα.
The Development of Improved Therapeutics through a Glycan- “Designer” Approach
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Knowing that the benefit of glycosylation serves tailored immunogenicity and activity of antimicrobial peptides, there is need for a fast, cheap, and reliable technique to attach glycan at the precise location. For this purpose, many techniques have been developed, including recombinant Protein–glycan coupling technology (PGCT) and recently published similar technique for O-linked glycan attachment, so-called site-directed glycosylation. The site-directed glycosylation similarly to PGCT uses E. coli as a host. However, it consists of two steps: Introduction of O-linked N-Acetylglucosamine (O-Glc-NAc) priming monosaccharide on the target protein/peptide followed by a ligation of eukaryotic N-glycan onto the O-GlcNAc (Wu et al., 2016). The first step requires catalyzing enzyme oligosaccharyltransferase (OGT) that transfers O-linked GlcNAc onto the accessible Serine/Threonine residue of a protein through β-linkage. In the method of site directed glycosylation both protein and the OGTase are expressed on separate plasmids in vivo E. coli. In the second step of transferring the N-glycan onto the primed protein researchers used an optimized enzyme endo-β-N-acetylglucosaminidase (Endo MN175Q) (Wu et al., 2016; Umekawa et al., 2010), which showed to have promiscuous substrate tolerance for GlcNAc-O-linked peptides/proteins in addition to its GlcNAc-N -linked natural preference. The efficiency of the O-linked substrate transfer was much lower (21%) then the N-linked one (73%) nonetheless with future improvements and optimizations this technique could be viable for tailoring glycans of interest to a monosaccharide primed protein.
PMM2-CDG (Congenital disorders of glycosylation, type Ia)
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
Among the most used tests for the diagnosis of this condition is the isoelectric focusing of serum transferrin, which reveals the characteristic CDG type I pattern due to the loss of complete N-glycan chains that lead to an increased amount of di- and asialotransferrin at the expense of tetrasialotransferrin [21, 22]. Half of this glycoprotein is found to lack two or four of its terminal sialic acid moieties. The normal transferrin of serum is predominantly tetrasialotransferrin, and there are small amounts of mono-, di-, tri-, penta-, and hexa-sialotransferrins; in the disease state, loss of negatively charged sialic acid causes a cathodal shift. Abnormal transferrin is also present in liver and CSF. Qualitative diagnosis is made by isoelectric focusing and immunofixation of transferrin. Quantitative determination of carbohydrate-deficient transferrin indicated an approximately ten-fold elevation of cathodal transferrin forms [21]. Electrophoresis reveals low molecular weight isoforms of many serum glycoproteins, including α-1 antitrypsin [22, 23]. The diagnostic accuracy may be improved using isoelectric focusing of α-1 antitrypsin and α-1 antichymotrypsin [23], and methodologies such as high-performance liquid chromatography (HPLC) and capillary zone electrophoresis [24] may be better suited to automation. The feasibility of tandem mass spectrometry has been demonstrated to elucidate the glycosylation of transferrin [25], an approach which allows for quantitative results and which offers the specificity to detect variant forms with more subtle differences in glycan processing. In contrast to fibroblasts from healthy controls, which produce mainly Glc3Man9GlcNAc2 oligosaccharides linked to the dolichol carrier, fibroblasts of PMM2-CDG patients show an accumulation of shortened dolichol-linked oligosaccharides such as Man3GlcNAc2 and Man5GlcNAc2. These represent unsuitable substrates for the oligosaccharyltransferase complex, thereby leading to the loss of oligosaccharide side chains linked to glycoproteins [26].The fundamental defect is in the synthesis and transfer of nascent dolichol-linked oligosaccharide precursors, and incorporation of labeled mannose into glycoproteins and the dolichol-linked oligosaccharide precursor is also shown to be deficient [27]. The abnormality in lipid-linked oligosaccharide biosynthesis could lead to failure to glycosylate sites on proteins and to abnormalities in glycoprotein processing or function. Furthermore, there is probably a cellular response to unfolded proteins that plays a part in the pathogenesis in this class of disease [28].
Molecular engineering tools for the development of vaccines against infectious diseases: current status and future directions
Published in Expert Review of Vaccines, 2023
Wenhui Xue, Tingting Li, Ying Gu, Shaowei Li, Ningshao Xia
Some glycoconjugate vaccines based on PGCT production have entered clinical trials for review, including O-antigen conjugates of type I Shigella dysenteriae, Shigella flexneri 2a, and uropathogenic pathogenic E. coli [121–123]. However, the efficient expression of capsule polysaccharides relies on specific bacterial strains due to their diversity and complexity. The coupling efficiency of complex polysaccharides with oligosaccharyltransferase is significantly influenced by their compatibility. Additionally, only one structure of OST (PglB) has been reported, and the relationship between its structure and glycosylation ability remains only partially understood [124]. Factors such as the protein carrier, the site of glycan coupling, and polymer length directly impact vaccine efficacy. These concerns pose challenges to the development of PGCT-based vaccines, emphasizing the need for further research to address these limitations [118].
The crosstalk between DNA damage response components and DNA-sensing innate immune signaling pathways
Published in International Reviews of Immunology, 2022
Feng Lin, Yan-Dong Tang, Chunfu Zheng
TREX1 (the three prime repair exonuclease 1) is the major exonuclease in the mammalian cytoplasm, and it degrades both ssDNA and dsDNA [46]. The cytoplasmic DNA clearing depends on the 3′→5′ DNA exonucleolytic function of TREX1, preventing cell-intrinsic inappropriate autoimmunity initiation [47,48]. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome and systemic lupus erythematosus in humans [49,50]. Oxidative DNA damage confers resistance to cytosolic nuclease TREX1 degradation and potentiates the STING-dependent innate immune signaling pathway [51]. Mutations that disrupt the mouse’s TREX1 exonuclease activity leads to the accumulation of self-DNA in the cytosol, which activates the cGAS-STING-mediated IFN-I response and systemic inflammation [52,53]. Another study finds that TREX1 C-terminus is the key to suppress immune activation by interacting with the ER oligosaccharyltransferase and stabilizing its catalytic integrity [54]. Thus, the nuclease activity of TREX1 helps cells clear the self-and foreign DNA in the cytosol, thereby preventing the unwarranted initiation of autoimmune signaling.
Improving protein glycan coupling technology (PGCT) for glycoconjugate vaccine production
Published in Expert Review of Vaccines, 2020
Jennifer Mhairi Dow, Marta Mauri, Timothy Alexander Scott, Brendan William Wren
Although originally thought to be the exclusive property of eukaryotes, it is now known that glycoproteins, and hence protein glycosylation systems are found in all domains of life [26]. The first evidence of a dedicated general protein glycosylation system in bacteria was found in the gastrointestinal pathogen Campylobacter jejuni [27]. The genetic locus responsible was designated pgl for protein glycosylation, and its existence was later confirmed by genomic sequencing of C. jejuni strain NCTC 11,168 in 2000 [28]. This system was shown to be an N-linked system, meaning the glycan is covalently bound to the amide nitrogen atom of the amide group of an asparagine (Asn, N) residue found within the consensus acceptor sequon D/EXNYS/T (where X and Y are any amino acid except proline) of a protein or peptide [29,30]. The most significant finding from this N-linked glycosylation system was the discovery of the oligosaccharyltransferase (OST) PglB, the enzyme responsible for coupling glycans to acceptor proteins (discussed in detail in Section 2.1.1) [31].