Improved sample preparation for CE-LIF analysis of plant N-glycans

In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the in-gel release method and N-glycan analysis on a multicapillary DNA sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells

GlyConnect-Ugi : site-selective, multi-component glycoprotein conjugations through GlycoDelete expressed glycans

Recently, the GlyConnect-oxime (GC) protein conjugation strategy was developed to provide a site-selective glycan-based conjugation strategy as an extension to the in-house developed GlycoDelete (GD) technology. GD gives access to glycoproteins with single GlcNAc, LacNAc, or LacNAc-Sia type glycans on their N-glycosylation sites. We have previously shown that these glycans provide a unique handle for site-selective conjugation as they provide a short, homogeneous and hydrophilic link to the protein backbone. GC focused on the use of chemical and chemo-enzymatic pathways for conjugation of a single molecule of interest via oxime formation or reductive amination. In the current work, we explore multicomponent reactions (MCR), namely Ugi and Passerini reactions, for GlycoDelete glycan directed, site-specific protein conjugation (MC-GC). The use of the Ugi and Passerini multicomponent reactions holds the potential of introducing multiple groups of interest in a single reaction step while creating a hydrophilic peptide-like linker

GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy—which we call GlycoDelete—that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian protins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions

GlycoVHH : optimal sites for introducing N-glycans on the camelid VHH antibody scaffold and use for macrophage delivery

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man(5)GlcNAc(2)-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man(5)GlcNAc(2) N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology

Using GlycoDelete to produce proteins lacking plant-specific N-glycan modification in seeds

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