Immobilized enzyme cascade for targeted glycosylation

Abstract

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation

“…We therefore sought to produce one of these trisaccharides using a completely in vitro enzymatic approach. LgtB, a glycosyltransferase from Neisseria meningitidis, has been shown to install galactose onto GlcNAc in a β-1,4 linkage, and PdST6, a glycosyltransferase from Photobacterium damselae, has been shown to install sialic acid in an α-2,6 linkage. ,, We incorporated these enzymes and the appropriate nucleotide-activated sugar donors, uridine diphosphate α-D-galactose (UDP- galactose) and cytidine monophosphate sialic acid (CMP-sialic acid), into our reaction solution to perform elaboration to a sialylated N -acetyllactosamine (sialyl-LacNAc) structure.…”

“…Our method could also be interfaced with other recent work developing in vitro, bead-based enzyme immobilization strategies for building glycosylation pathways. 22 Future work will focus on expanding the capabilities of our platform for producing therapeutically relevant glycoproteins. For example, our proof-of-concept work presented here demonstrates efficient glycosylation of a model sfGFP construct using a C-terminal sequon.…”

“…For example, our one-step GlcNAcylation method could simplify chemoenzymatic transglycosylation approaches that utilize a mutated endoglycosidase (ENGase) to transfer chemically synthesized glycan polymers onto proteins with a single GlcNAc residue, , by removing the requirement of time-intensive mammalian cell culture and glycan trimming steps to produce the single GlcNAc residue. Our method could also be interfaced with other recent work developing in vitro , bead-based enzyme immobilization strategies for building glycosylation pathways …”

“…In comparison to in vivo –based approaches, the modularity and open reaction environment of cell-free or in vitro glycosylation (IVG) systems could aid in the development of new glycoprotein production methods. Indeed, recent work in the field has sought to create routes to glycosylated proteins and peptides using glycosyltransferases heterologously expressed in living Escherichia coli − or in cell-free protein synthesis (CFPS) − and subsequently assembled in IVG reactions. To date, there have been several attempts to use these systems to create glycoproteins harboring a single N -GlcNAc residue, which differentiate from the strategy presented in this work through the use of (i) multienzyme methods with sugar donors that are not commercially available, (ii) synthetic GlcNAc donor substrates composed of GlcNAc coupled to chemically synthesized polyisoprenol sugar pyrophosphates, , (iii) cell-based biosynthesis of larger bacterial or eukaryotic N -linked glycan structures that must be subsequently trimmed in vitro by a glycosidase, , or (iv) complicated multistep chemical synthesis routes − to produce the priming GlcNAc residue.…”

Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis

“…The immobilization of GTs on a specific carrier could eliminate this problem by enabling not only the reuse of the enzyme but also a simpler product isolation. Only a few GTs have been immobilized for their use in glycan synthesis, − making it all the more important to take this step to enable more economical synthesis of glycans. The superordinate goal is not only the reuse of enzymes but also the establishment of a fully automated and scalable synthesis of customized glycans. , …”