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  • br Glycoprotein Production br Strategies to Obtain Glycoprot


    Glycoprotein Production
    Strategies to Obtain Glycoprotein Compositional Homogeneity Glycoproteins are modified by glycosyltransferases and glycosylhydrolases in the ER and Golgi; however, the degree of remodeling for each glycan on each protein is not explicitly defined by a template (Moremen et al., 2012). This template-independent synthesis leads to potentially enormous compositional variability that is evident as a smear in an SDS-PAGE gel instead of a single, tight band. This section contains a description of multiple methods available to obtain homogeneity that manipulate N-glycans during protein expression, postpurification, or a combination of both. These methods have advanced glycoprotein studies, however, it is not always clear what types of glycans are found on glycoproteins in the human body, and proteins expressed by HEK293F cells do not necessarily contain the correct types of N-glycans, thus, caution is suggested (Patel, Roberts, Subedi, & Barb, 2018). Another aspect that is not addressed by any of these techniques is the accessibility of the N-glycosylation site to the oligosaccharyltransferase. Asparagine residues in the extreme N-terminus or within 50 residues of the C-terminus may not be efficiently modified (Kelleher & Gilmore, 2006).
    Compositional Analysis of Glycoproteins One critical aspect of glycan remodeling is assessing compositional heterogeneity arising from incomplete remodeling reactions in vivo and in vitro. Though many techniques are available, we use predominantly mass spectrometry-based methods due to rapidity and precision. In addition to confirming sample homogeneity, N-glycan processing during recombinant protein expression is a useful reporter of glycan accessibility. Analyses of N-glycan composition before enzymatic remodeling has the potential to directly support observations made by NMR spectroscopy (Subedi, Hanson, & Barb, 2014). For example, N-glycans that stabilize protein structure often form van Der Waals contacts with Nicotinamide australia at the protein surface. These interactions restrict N-glycan processing and restrict enzymatic N-glycan remodeling in vivo and in vitro.
    Solution NMR of Labeled Glycoproteins NMR spectra of isotope-labeled glycoproteins are collected with the same pulse sequences applied to most [13C, 15N]-labeled proteins. Here we describe considerations for NMR analyses of glycoproteins with labeled backbone residues and labeled carbohydrates.
    Introduction The occurrence of acquired drug resistance severely impairs the successful treatment of cancer with chemotherapeutic drugs [1,2]. Cancer cells can develop resistance to anti-cancer drugs via two different mechanisms. Firstly, the intracellular drug concentration can be decreased, e.g. by enhancing cellular drug efflux. Secondly, intracellular signaling pathways can be altered which can cause drug resistance even though adequate intracellular drug levels have been achieved [3]. Cancer cells can extrude chemotherapeutic drugs by hijacking cellular systems used in non-malignant cells for extrusion of metabolic products and detoxification [4]. P-glycoprotein was the first member of the ATP-binding cassette sub-family B (ABC) transporter family to be discovered in cancer [[5], [6], [7], [8]]. Many tumor cells express p-glycoprotein, and some carry genetic amplifications of the underlying gene MDR1 [9,10]. P-glycoprotein is localized in the plasma membrane and can pump substrates out of the cell in an ATP-dependent manner [11]. It has a broad affinity to various substrates including chemotherapeutic agents like DOXO, ETO, VCR and glucocorticoids [12]. Hence, chemotherapy-resistant tumors that display increased p-glycoprotein expression can acquire cross-resistance to multiple other, structurally unrelated drugs, which leads to MDR. Inhibition of p-glycoprotein or similar ABC-transporters using small-molecule inhibitors that modulate drug efflux has long been discussed as a promising strategy to combat MDR in cancer [13]. Several pharmacological inhibitors have been developed, including first-generation compounds (e.g. Verapamil [14]), second-generation compounds (e.g. PSC833/Valspodar [15]) and third-generation compounds (e.g. Tariquidar [16]). However, the results of clinical trials aiming to use p-glycoprotein inhibitors to reverse drug resistance have overall been disappointing [17], which is mainly due to the highly toxic effects exerted by these inhibitors on normal cells, either directly or indirectly by decreasing the elimination of the anti-cancer drugs and hence increasing their toxicity on normal tissues when administered in combination [18]. Toxic effects on normal tissues could be overcome by cancer-specific drug delivery systems, which are currently under development.