A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans

Abstract

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Molecular features and predictive models identify the most lethal subtype and a therapeutic target for osteosarcoma

“…Cholesterol accumulation was facilitated by cholesterol biosynthesis and cellular uptake of LDL-cholesterol, while cholesterol consumption was mainly regulated by cholesterol esterification and efflux ( 45 ). When cholesterol accumulates to a high level, cells relieve cellular stress through esterification and efflux ( 46 ). In S-IV patients, mediators involved in cholesterol biosynthesis (HMGCS, HMGCR, SQLE) and cholesterol consumption (ACAT1, ACAT2, ABCA1, ABCG1) were highly expressed, while regulators promoting cellular uptake of LDL-cholesterol (LDLR, Niemann-Pick C proteins including NPC1 and NPC2) were not active.…”

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

“…Nevertheless, the scope of several reported examples [ 25 , 26 , 27 , 28 ] is limited to the involvement of one or two glycosyltransferases with or without glycoside hydrolases. The utilization of multiple carbohydrate-active enzymes including glycoside hydrolases and glycosyltransferases for glycoprotein in vitro N-glycan processing has been traditionally underexplored, but has shown a high potential in several recent examples [ 23 , 29 ]. In general, glycosyltransferases that are expressed in mammalian [ 24 , 27 ] or insect cells [ 25 ] have been commonly used for glycoprotein N-glycan engineering due to the challenges in obtaining their active forms from common E. coli expression systems [ 29 , 30 ].…”

“…The utilization of multiple carbohydrate-active enzymes including glycoside hydrolases and glycosyltransferases for glycoprotein in vitro N-glycan processing has been traditionally underexplored, but has shown a high potential in several recent examples [ 23 , 29 ]. In general, glycosyltransferases that are expressed in mammalian [ 24 , 27 ] or insect cells [ 25 ] have been commonly used for glycoprotein N-glycan engineering due to the challenges in obtaining their active forms from common E. coli expression systems [ 29 , 30 ]. As many of these enzymes expressed in eukaryotic cells are N-glycosylated glycoproteins themselves, their N-glycans may complicate N-glycan analysis of target glycoproteins, a potential problem that deserves consideration and investigation, but has not been paid sufficient attention.…”

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry

“…One strategy to overcome these challenges is cell-free expression (CFE), which harnesses biological machinery to enable high-yielding transcription and translation outside of the living cell (Carlson et al 2012; Silverman et al 2020). The modular and open CFE reaction environment allows for manipulation of expression conditions, enabling production of complex products including proteins containing disulfide bonds (Goerke and Swartz 2008; Dopp and Reuel 2020), membrane proteins (Matthies et al 2011; Kruyer et al 2021), and glycosylated proteins (Kightlinger et al 2019; Hershewe et al 2021; Stark et al 2021; Jaroentomeechai et al 2022). CFE is also scalable from the nanoliter to liter scale (Zawada et al 2011), allowing for small-scale parallel expression of many proteins simply by switching out the template DNA added to the reaction, and accelerating protein screening (Hunt et al 2021; Hunt et al 2022).…”