Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms.

Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial ß1,4 galactosyltransferase from Neiserria meningitidis and human ß1,4 galactosyltransferase IV（B4GALT4）, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.

Synthetic Antibody-Rhamnose Cluster Conjugates Show Potent Complement-Dependent Cell Killing by Recruiting Natural Antibodies.

Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement-dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well-defined conjugates of a monoclonal antibody with a rhamnose- and an αGal trisaccharide-cluster to recruit natural anti-rhamnose and anti-αGal antibodies, respectively, to enhance the CDC-dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc-glycan remodeling method that includes site-specific chemoenzymatic Fc-glycan functionalization and subsequent click conjugation of synthetic rhamnose- and αGal trisaccharide-cluster to provide the respective homogeneous antibody conjugates. Cell-based assays indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody-αGal trisaccharide cluster conjugates for CDC effects.

General and Robust Chemoenzymatic Method for Glycan-Mediated Site-Specific Labeling and Conjugation of Antibodies: Facile Synthesis of Homogeneous Antibody-Drug Conjugates.

Site-specific labeling and conjugation of antibodies are highly desirable for fundamental research and for developing more efficient diagnostic and therapeutic methods. We report here a general and robust chemoenzymatic method that permits a one-pot site-specific functionalization of antibodies. A series of selectively modified disaccharide oxazoline derivatives were designed, synthesized, and evaluated as donor substrates of different endoglycosidases for antibody Fc glycan remodeling. We found that among several endoglycosidases tested, wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody-drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody-drug conjugation but also for cell labeling, imaging, and diagnosis.

A facile chemoenzymatic synthesis of SARS-CoV-2 glycopeptides for probing glycosylation functions.

Glycosylation plays important roles in SARS-CoV-2 infection. We describe here a facile chemoenzymatic synthesis of core-fucosylated N-glycopeptides derived from the SARS-CoV-2 Spike protein and their binding with glycan-dependent neutralizing antibody S309 and human lectin CLEC4G. The synthetic glycopeptides provide tools for further functional characterization of viral glycosylation.

Comparative studies on the substrate specificity and defucosylation activity of three α-l-fucosidases using synthetic fucosylated glycopeptides and glycoproteins as substrates.

Core fucosylation is the attachment of an α-1,6-fucose moiety to the innermost N-acetyl glucosamine (GlcNAc) in N-glycans in mammalian systems. It plays a pivotal role in modulating the structural and biological functions of glycoproteins including therapeutic antibodies. Yet, few α-l-fucosidases appear to be capable of removing core fucose from intact glycoproteins. This paper describes a comparative study of the substrate specificity and relative activity of the human α-l-fucosidase (FucA1) and two bacterial α-l-fucosidases, the AlfC from Lactobacillus casei and the BfFuc from Bacteroides fragilis. This study was enabled by the synthesis of an array of structurally well-defined core-fucosylated substrates, including core-fucosylated N-glycopeptides and a few antibody glycoforms. It was found that AlfC and BfFuc could not remove core fucose from intact full-length N-glycopeptides or N-glycoproteins but could hydrolyze only the truncated Fucα1,6GlcNAc-peptide substrates. In contrast, the human α-l-fucosidase (FucA1) showed low activity on truncated Fucα1,6GlcNAc substrates but was able to remove core fucose from intact and full-length core-fucosylated N-glycopeptides and N-glycoproteins. In addition, it was found that FucA1 was the only α-l-fucosidase that showed low but apparent activity to remove core fucose from intact IgG antibodies. The ability of FucA1 to defucosylate intact monoclonal antibodies reveals an opportunity to evolve the human α-l-fucosidase for direct enzymatic defucosylation of therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity.

Protection against SARS-CoV-2 infection by a mucosal vaccine in rhesus macaques.

Effective SARS-CoV-2 vaccines are urgently needed. Although most vaccine strategies have focused on systemic immunization, here we compared the protective efficacy of 2 adjuvanted subunit vaccines with spike protein S1: an intramuscularly primed/boosted vaccine and an intramuscularly primed/intranasally boosted mucosal vaccine in rhesus macaques. The intramuscular-alum-only vaccine induced robust binding and neutralizing antibody and persistent cellular immunity systemically and mucosally, whereas intranasal boosting with nanoparticles, including IL-15 and TLR agonists, elicited weaker T cell and Ab responses but higher dimeric IgA and IFN-α. Nevertheless, following SARS-CoV-2 challenge, neither group showed detectable subgenomic RNA in upper or lower respiratory tracts versus naive controls, indicating full protection against viral replication. Although mucosal and systemic protective mechanisms may differ, results demonstrate both vaccines can protect against respiratory SARS-CoV-2 exposure. In summary, we have demonstrated that the mucosal vaccine was safe after multiple doses and cleared the input virus more efficiently in the nasal cavity and thus may act as a potent complementary reinforcing boost for conventional systemic vaccines to provide overall better protection.

Exploring a combined Escherichia coli-based glycosylation and in vitro transglycosylation approach for expression of glycosylated interferon alpha.

The conventional use of E. coli system for protein expression is limited to non-glycosylated proteins. While yeast, insect and mammalian systems are available to produce heterologous glycoproteins, developing an engineered E. coli-based glycosylation platform will provide a faster, more economical, and more convenient alternative. In this work, we present a two-step approach for production of a homogeneously glycosylated eukaryotic protein using the E. coli expression system. Human interferon α-2b (IFNα) is used as a model protein to illustrate this glycosylation scheme. In the first step, the N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) is co-expressed for in vivo transfer of a glucose residue to IFNα at an NX(S/T) N-glycosylation sequon. Several E. coli systems were examined to evaluate the efficiency of IFNα N-glucosylation. In the second step, the N-glucosylated protein is efficiently elaborated with biantennary sialylated complex-type N-glycan using an in vitro chemoenzymatic method. The N-glycosylated IFNα product was found to be biologically active and displayed significantly improved proteolytic stability. This work presents a feasible E. coli-based glycosylation machinery for producing therapeutic eukaryotic glycoproteins.

Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases.

Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to site-specifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.