Metabolic Glyco-Engineering in Eukaryotic Cells and Selected Applications

Abstract

By metabolic glyco-engineering cellular glycoconjugates are modified through the incorporation of synthetic monosaccharides which are usually analogues of naturally present sugars. In order to get incorporated, the monosaccharides need to enter the cytoplasm and to be substrates for the enzymes necessary for their transformation into activated sugars, most often nucleotide sugars. These have to be substrates for glycosyltransferases which finally catalyze their incorporation into glycans. Such pathways are difficult to reconstitute in vitro and therefore new monosaccharide analogues have to be tested in tissue culture for their suitability in metabolic glyco-engineering. For this, glycosylation mutants are the most appropriate since they are unable to synthesize specific glycans but through the introduction of the monosaccharide analogues they may express some glycans at the cell surface with the unnatural sugar incorporated. The presence of those glycans can be easily and quantitatively detected by lectin binding or by chemical methods identifying specific sugars. Monosaccharide analogues can also block the pathways leading to sugar incorporation, thus inhibiting the synthesis of glycan structures which is also easily detectable at the cell surface by lectin labeling. The most useful and most frequently employed application of metabolic glyco-engineering is the introduction of reactive groups which can undergo bio-orthogonal click reactions for the efficient labeling of glycans at the surface of live cells.

Transforms of Cell Surface Glycoproteins Charge Influences Tumor Cell Metastasis via Atypically Inhibiting Epithelial-Mesenchymal Transition Including Matrix Metalloproteinases and Cell Junctions

“…37−39 Although Ac4ManNAc can be converted intracellularly to sialic acid with azide groups, considering that Ac4ManNAc is poorly watersoluble and significant cytotoxicity, we chose 9Az-Sia for glycolabeling. 40,41 Different concentrations of 9Az-Sia (ranging from 0−400 μM) had little effect on the viability of MDA-MB-231 cells, confirming the bio-orthogonality of 9Az-Sia (Figure 2B). To confirm the validity of MGL, we selected the fluorescent click chemistry labeling with DBCO-PEG4-biotin and FITC-avidin.…”

“…As shown in Figure A, we verified the mainly groups on 9Az-Sia by infrared spectroscopy, especially the characteristic band of the azide stretching vibration located near 2110 cm –1 . The principles of bio-orthogonal chemistry proposed by Bertozzi should be followed for metabolic glycolabeling and glycoengineering, which means that chemical reactions to label the cell or organism must not interfere with other chemical reactions in the biological system. , The results of previous studies have shown that sialic acid maintains normal biological activity when the group on C5 or C9 of sialic acid is replaced by an azide group, so 9Az-Sia and Ac4ManNAc are commonly used for sialic acid glycan labeling. − Although Ac4ManNAc can be converted intracellularly to sialic acid with azide groups, considering that Ac4ManNAc is poorly water-soluble and significant cytotoxicity, we chose 9Az-Sia for glycolabeling. , Different concentrations of 9Az-Sia (ranging from 0–400 μM) had little effect on the viability of MDA-MB-231 cells, confirming the bio-orthogonality of 9Az-Sia (Figure B). To confirm the validity of MGL, we selected the fluorescent click chemistry labeling with DBCO-PEG4-biotin and FITC-avidin. , The results of flow cytometry and fluorescence confocal microscopy show that 9Az-Sia can be translocated to the cell membrane surface and show that click chemistry can be used to alter the charge of glycoproteins (Figure C,D).…”

Protocol Considerations for In Vitro Metabolic Glycoengineering of Non‐Natural Glycans

“…Accordingly, MGE technology holds great potential for manipulating basic cellular functions for medical applications such as tissue engineering, cancer therapy, and carbohydrate-based medicine. Here (as well as in other publications, e.g., Piller et al, 2015;Wratil & Horstkorte, 2017), the general steps and protocols for characterization of novel sugar analogs used for MGE are described. Critical parameters for consideration and common problems related to these experiments are discussed below.…”