Systematic Preparation of a 66-IgG Library with Symmetric and Asymmetric Homogeneous Glycans and Their Functional Evaluation.

Immunoglobulin G (IgG) antibodies possess a conserved N-glycosylation site in the Fc domain. In FcγRIIIa affinity column chromatography, unglycosylated, hemiglycosylated, and fully glycosylated IgG retention times differ considerably. Using retention-time differences, 66 different trastuzumab antibodies with symmetric and asymmetric homogeneous glycans were prepared systematically, substantially expanding the scope of IgGs with homogeneous glycans. Using the prepared trastuzumab with homogeneous glycans, thermal stability and antibody-dependent cellular cytotoxicity were investigated. In some glycan series, a directly proportional relationship was observed between the thermal unfolding temperature (Tm) and the calorimetric unfolding heat (ΔHcal). Antibody function could be deduced from the combination of a pair of glycans in an intact form. Controlling glycan structure through the combination of a pair of glycans permits the precise tuning of stability and effector functions of IgG. Overall, our technology can be used to investigate the effects of glycans on antibody functions.

Characterization of Antibody Products Obtained through Enzymatic and Nonenzymatic Glycosylation Reactions with a Glycan Oxazoline and Preparation of a Homogeneous Antibody-Drug Conjugate via Fc N-Glycan.

Glycan engineering of antibodies has received considerable attention. Although various endo-ß- N-acetylglucosaminidase mutants have been developed for glycan remodeling, a side reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and nonenzymatic reactions with the aim of maximizing the efficiency of the glycosylation reaction with fewer side products. The reactions were monitored by an ultraperformance liquid chromatography system using an amide-based wide-pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through a combination of an azide-carrying glycan transfer reaction under optimized conditions and a bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with a drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.

Characterization of novel endo-ß-N-acetylglucosaminidases from intestinal Barnesiella intestinihominis that hydrolyze multi-branched complex-type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Author Correction: Characterization of novel endo-ß-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of novel endo-ß-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

Endo-ß-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3-2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.