Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein–protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

Glycosylation of asparagine residues is one of the most abundant and structurally complex protein posttranslational modifications (1, 2) and occurs in all domains of life (3). Owing to their relatively large size and hydrophilicity or simply their presence at definite locations, asparagine-linked (N-linked) glycans can significantly alter protein properties including biological activity, chemical solubility, folding and stability, immunogenicity, and serum half-life (4, 5). Hence, glycosylation effectively increases the diversity of the proteome by enriching the repertoire of protein characteristics beyond that dictated by the 20 canonical amino acids. For example, accumulating evidence indicates that the immune system diversifies the repertoire of antigen specificities by exclusively targeting the antigen-binding sites of immunoglobulins (IgGs) with posttranslational modifications, in particular N-linked glycosylation (6). Moreover, the profound effect of glycans on proteins has prompted widespread glycoengineering efforts to rationally manipulate key glycosylation parameters (e.g., glycan size and structural composition and glycosite location and occupancy) as a means to optimize protein traits for a range of different industrial and therapeutic applications (7–10).Despite some notable successes, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is currently limited by our inability to predict which sites within a protein are glycosylatable and how glycosylation at permissive sites will affect protein structure and function. Indeed, a deeper understanding of the design rules (i.e., how glycans influence the biological and biophysical properties of a protein) represents a grand challenge for the glycoprotein engineering field. To this end, computational approaches have enabled in silico exploration of glycosylation-induced effects on protein folding and stability (11, 12); however, these involve a trade-off between molecular detail and glycoprotein size, with full-atomistic molecular dynamics simulations typically limited to only short glycopeptides or protein domains (11). To experimentally probe the consequences of glycosylation ideally requires access to large collections of chemically defined glycoproteins in sufficient quantities for characterization (13). Mammalian cells represent an obvious choice to source proteins with both natural and naïve glycosites. However, because of the time-consuming, low-throughput nature of gene transfection and culturing of mammalian cells, studies using mammalian cell-based expression systems have typically only investigated a small number of designs (∼15 or fewer) (14–17), with rare exceptions such as the tour de force study by Elliott et al. (18). In addition, the intrinsic variability with respect to the glycan structure at a given site (microheterogeneity) can be unpredictable and difficult to control in mammalian expression systems. Another option is chemical synthesis, which can furnish structurally uniform glycopeptides for investigating the local effects of N-linked glycans on peptide conformation (19). While total chemical synthesis remains challenging for full-length proteins, advances in expressed protein ligation (EPL) have opened the door to convergent assembly of chemically synthesized glycopeptides with recombinantly expressed protein domains to form larger glycoproteins bearing complex N-glycans installed at discrete sites (20, 21). Using this technology, Imperiali and coworkers created a panel of seven site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the disaccharide N,N''-diacetylchitobiose (GlcNAc₂) and assessed the kinetic and thermodynamic consequences of glycan installation at defined locations (22). Unfortunately, EPL is a technically demanding procedure, requiring manual construction of each individual glycoprotein, which effectively limits the number of testable glycosite designs to just a small handful.To move beyond these “one-glycosite-at-a-time” methods for supplying glycoproteins, herein we describe a scalable technique called shotgun scanning glycomutagenesis (SSGM) that involves design and construction of combinatorial acceptor protein libraries in which 1) each member of the library carries a single N-glycosite “mutation” introduced at a defined position along the protein backbone and 2) the complete ensemble of glycan acceptor sites (sequons) in the library effectively covers every possible position in the target protein (Fig. 1). The resulting SSGM libraries are expressed using N-glycosylation-competent bacteria in the context of glycoSNAP (glycosylation of secreted N-linked acceptor proteins), a versatile high-throughput screen based on extracellular secretion of glycosylated proteins (23). Using this glycoprotein engineering tool, we constructed and screened SSGM libraries corresponding to three model proteins: bacterial immunity protein Im7, bovine pancreatic ribonuclease A (RNase A), and a human single-chain variable fragment antibody specific for HER2 (scFv-HER2). Our results revealed that installation of N-glycans was tolerated at a large number of positions and in all types of secondary structure, with relatively high N-glycosylation efficiency in the majority of cases. For many of these glycoproteins, the presence of N-glycans at naïve sites had a measurable effect on protein stability and/or activity in a manner that depended on the precise location of the modification. Taken together, these findings demonstrate the ability of the SSGM method to yield large collections of discretely modified neoglycoproteins that collectively reveal glycosylatable sites and provide insight on the influence that site-specific N-glycan installation has on structural and/or functional properties.Open in a separate windowFig. 1.Constructing neoglycoproteins by SSGM. Schematic of SSGM, a glycoprotein engineering method based on combinatorial protein libraries in which glycosylation “sequon walking” is used to introduce an acceptor site at every possible position along a protein backbone. Note that the multiresidue nature of a sequon (e.g., N-X-S/T or D/E-X₁-N-X₂-S/T, where X, X₁, X₂ ≠ P) necessitates insertion or replacement of up to five additional amino acid substitutions at each position. The resulting library is expressed in glycoengineered bacteria, providing an opportunity for each library member to be expressed and glycosylated in a manner that is compatible with high-throughput screening via glycoSNAP to interrogate the glycosylation phenotype of individual variants. By integrating expressed SSGM libraries with multiplexable assays, the biochemical and biophysical properties of each neoglycoprotein can be individually interrogated. Depicted is the engineered C. jejuni GalNAc₅(Glc)GlcNAc heptasaccharide with reducing end GlcNAc (blue square) followed by five GalNAc residues (yellow squares) and a branching glucose (blue circle). Structure drawn according to symbol nomenclature for glycans (SNFG; https://www.ncbi.nlm.nih.gov/glycans/snfg.html).