Mammalian cell-based production of glycans, glycopeptides and glycomodules

Abstract

Access to defined glycans and glycoconjugates is pivotal for discovery, dissection, and harnessing of a range of biological functions orchestrated by cellular glycosylation processes and the glycome. We previously employed genetic glycoengineering by nuclease-based gene editing to develop sustainable production of designer glycoprotein therapeutics and cell-based glycan arrays that display glycans in their natural context at the cell surface. However, access to human glycans in formats and quantities that allow structural studies of molecular interactions and use of glycans in biomedical applications currently rely on chemical and chemoenzymatic syntheses associated with considerable labor, waste, and costs. Here, we develop a sustainable and scalable method for production of glycans in glycoengineered mammalian cells by employing secreted Glycocarriers with repeat glycosylation acceptor sequence motifs for different glycans. The Glycocarrier technology provides a flexible production platform for glycans in different formats, including oligosaccharides, glycopeptides, and multimeric glycomodules, and offers wide opportunities for use in bioassays and biomedical applications.

Fig. 1. Graphic overview of the Glycocarrier technology.

a A library of glycoengineered cells generated by combinatorial knock-out (KO) and knock-in (KI) of glycosyltransferase genes is used for recombinant expression of secreted Glycocarriers containing glycomodules designed to be glycosylated with different types of glycans. b Glycocarriers are designed to contain a folded globular module (GFP or IgG Fc) for efficient secretion, polyhistidine and site-specific biotinylation (AVI) tags for purification, and a flexible glycomodule (~200 amino acids) designed with one or more short repeat acceptor substrate sequence motifs for glycosylation interspaced by Lys residues for tryptic digestion. Intact glycodomodules can be released from the globular domain using TEV protease digestion. c The Glycocarrier design provides opportunities for production of glycomodules with multimeric presentation of glycans as well as production of glycopeptides and glycans in amplified yields. Structures of glycans are shown with symbols drawn according to the Symbol Nomenclature for Glycans (SNFG) format. Parts of this figure were created in BioRender under agreement no. CR27EEXSTH.

Fig. 2. O-Glycocarriers for production of GalNAc-type O-glycans.

a Graphic overview of the human O-glycosylation pathway with glycosyltransferases assigned to key biosynthetic steps. b Intact MS analysis of TEV released O-glycomodules expressed in wild-type and cells glycoengineered as indicated. c LC-MS/MS analysis of the corresponding trypsin released glycopeptides. Spectra assigned “neu” indicate that analysis was performed after pretreatment with neuraminidase. P denotes nonglycosylated peptide.

Fig. 3. N-glycocarriers for production of N-glycans.

a Graphic overview of the human N-glycosylation pathway with glycosyltransferases assigned to key biosynthetic steps. b Comprehensive workflow for production and analysis of multimeric (10 N-glycosites of an 18-mer acceptor sequence LAGQALLVNSSQPWEALK) N-glycomodule, N-glycopeptides, and released N-glycans illustrated with the N-Glycocarrier expressed in CHO^{KO Mgat1} cells engineered to produce homogenous Man₅GlcNAc₂ N-glycan. Intact MS analysis of the TEV released N-glycomodule with and without endo-H treatment and LC-MS/MS analysis of the tryptic digested N-glycopeptides revealed complete N-glycan occupancy. PNGase F treatment of the N-glycomodule released homogeneous Man₅GlcNAc₂ N-glycans. Alternatively, extensive pronase digestion of the N-Glycocarrier produced N-glycans attached to a few amino acids (1-4) (upper right panel). c LC-MS/MS analysis of the tryptic released N-glycopeptides expressed in CHO^WT and CHO cells glycoengineered as indicated.

Fig. 4. GAG-Glycocarriers for production of glycosaminoglycans.

a Graphic overview of the human GAG biosynthesis pathway with glycosyltransferases assigned to key biosynthetic steps. b Design variants of GAG-Glycocarriers with three different acceptor peptide sequences. Monomeric human IgG-Fc was used as N-terminal globular domain, followed by 1 or 3 tandem repeats of GAG substrate sequences, separated by a TEV protease site, and with a C-terminal StrepII tag. c LC-MS/MS analysis of TEV and trypsin released glycopeptides from the three different GAG-Glycocarrier designs with single acceptor sequence motifs expressed in CHO^{KO B4galt7} to evaluate initiation efficiency (xylose incorporation). d SDS-PAGE Coomassie analysis of the purified GAG-glycocarrier 1 expressed in CHO^WT, CHO^{KO B4galt7}, CHO^{KO Extl2,3}, and CHO^{KO Csgalnac1/2/Chsy1} digested with heparinase (HepRI-III) and chondroitinase (ChABC) as indicated. Asterisk indicates non-specific contaminants. Results are the representation of three biological replications.

Fig. 5. Glycocarriers expand the glycoscience toolbox.

Glycocarriers produced in glycoengineered cells enable production of mammalian glycans in different formats for great diversity in applications. a Different presentation modes of glycans enable dissection of binding interactions with GBPs. b Glycocarriers provide scaffold mass that enables single molecule analytics, e.g., by mass photometry and LLPS. c Glycocarriers are tagged (GFP) and can easily be functionalized by chemical and metabolic methods for probing interactions and following cellular trafficking and biodistribution. d Glycocarriers facilitate synthesis and design of glycoproteins by supplying glycan and glycopeptide building unit. Parts of this figure were created in BioRender under agreement no. QB27EEYK24.