Exploiting metabolic glycoengineering to advance healthcare

Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the “glycan code”) and its application (e.g., biomanufacturing high-value glycomolecules on demand).

Section: Resultsmentioning

confidence: 99%

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

Jaroentomeechai

Taw

et al. 2020

“…Based on these considerations, this report establishes a set of sialoglycoproteins as novel targets for investigation into how glycosylation impacts their biological activity in both health and malignant disease. Consequently, this work augments growing efforts to translate metabolic glycoengineering into clinical healthcare (Agatemor et al, 2019). Although entirely speculative at this point, one way our 1,3,4-O-Bu 3 ManNAc-based approach can be envisioned to be deployed clinically is through emerging in vitro functional assays of a patient's living cancer cells (Kodack et al, 2017).…”

Section: Discussionmentioning

confidence: 98%

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression

Saeui

Cho²,

Dharmarha

et al. 2020

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Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu 3 ManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu 3 ManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu 3 ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (∼60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can Saeui et al. Flux-Based Control of Sialylation evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro-or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

“…In addition to the glycoengineering method of adjusting the expression and activity of enzymes involved in glycan biosynthesis and the method of increasing the number of glycosylation sites, another commonly used method is metabolic glycoengineering, a technique that was developed almost thirty years ago where protein glycosylation can be altered by changing the concentrations of monosaccharides or nucleotide sugars in the culture media (Bailey, 1991 ; Gramer et al, 2011 ; Buettner et al, 2018 ; Agatemor et al, 2019 ). A representative example is the study by Gu and Wang ( 1998 ) in which 20 mM of N-acetylaminomannose (ManNAc) was added to the culture media of CHO cells.…”

Section: Cell-based Protein Glycoengineeringmentioning

confidence: 99%

“…We will only touch upon a limited number of examples for each research direction. Interested readers may refer to more comprehensive reviews for detailed information (Bailey, 1991 ; Wright and Morrison, 1997 ; Saxon and Bertozzi, 2001 ; Bretthauer, 2003 ; Sinclair and Elliott, 2005 ; Hamilton and Gerngross, 2007 ; Beck et al, 2008 ; Beck and Reichert, 2012 ; Beckham et al, 2012 ; Baker et al, 2013 ; Merritt et al, 2013 ; Dicker and Strasser, 2015 ; Geisler et al, 2015 ; Greene et al, 2015 ; Buettner et al, 2018 ; Mimura et al, 2018 ; Montero-Morales and Steinkellner, 2018 ; Tejwani et al, 2018 ; Wang et al, 2018 , 2019 ; Yates et al, 2018 ; Agatemor et al, 2019 ; Harding and Feldman, 2019 ; Mastrangeli et al, 2019 ). In addition to glycoengineering using naturally occurring glycans and glycosidic linkages to improve the properties of proteins, there are many research efforts geared toward chemical and enzymatic synthesis of glycans, development of glycan-based vaccines and adjuvants, or using unnatural glycans and site-selective conjugation chemistry to achieve protein glycoengineering objectives.…”

Section: Introductionmentioning

confidence: 99%

Protein Glycoengineering: An Approach for Improving Protein Properties

Guan

et al. 2020

Natural proteins are an important source of therapeutic agents and industrial enzymes. While many of them have the potential to be used as highly effective medical treatments for a wide range of diseases or as catalysts for conversion of a range of molecules into important product types required by modern society, problems associated with poor biophysical and biological properties have limited their applications. Engineering proteins with reduced side-effects and/or improved biophysical and biological properties is therefore of great importance. As a common protein modification, glycosylation has the capacity to greatly influence these properties. Over the past three decades, research from many disciplines has established the importance of glycoengineering in overcoming the limitations of proteins. In this review, we will summarize the methods that have been used to glycoengineer proteins and briefly discuss some representative examples of these methods, with the goal of providing a general overview of this research area.