CD BioGlyco, as a company specializing in the field of glycobiology, has a huge library of glycan resources. We have constructed a Glycan Display Platform to provide our clients with Glyco-engineered Cell Construction Services from multiple perspectives. Here, we provide glycan engineering cell construction services based on the O-GalNAc pathway.
Due to the high complexity of glycosylation, we chose the HEK293 cell line as a cell construction platform because it can be used extensively for glycoproteins.
We construct cell lines with specific O-glycosylation features by adding and removing complex branches of O-glycosylation in the HEK293 cell line by the knock-in (KI)/knock-out (KO) technique of the glycosyltransferase gene. The positive or negative signals provided by the loss or gain of the KO/KI interaction of the glycosyltransferase gene serve to explain the structural features of the glycan and the glycoconjugates involved. For example, we utilize combinatorial CRISPR/Cas9 KO targeting to control GalNAc-type O-glycoproteins to display O-glycan features on the glycoconjugates. Alternatively, GalNAc-type O-glycans are displayed by eliminating the pathway-specific extension step of one or more glycoconjugates to differentially display the major types of glycoconjugates.
In addition, we perform an O-glycan analysis of total cell lysates to provide a site-specific analysis of the O-GalNAc glycoprotein reporter gene. The location and pattern of O-glycan modifications are determined by the peptide sequence (distribution of Ser/Thr residues) and the specificity of the available polypeptide GalNAc transferase that controls the initiation of O-glycosylation.
Across the board, we use RNA sequencing (RNA-seq) transcriptomics to detect the O-GalNAc capacity and structure of HEK293 cells. Flow cytometry is used to detect glycosyltransferase genes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to analyze O-glycan sites and structures.
Fig.1 O-GalNAc pathway-based glyco-engineered cell construction service. (CD BioGlyco)
Technology: Glycoengineering technique and homozygous cell model
Journal: Molecular Cell
IF: 16.0
Published: 2019
Results: General scaffolding and structure of glycoproteins can be predicted from a library of only 153 glycosyltransferase genes. Mapping of glycosylation pathways (N-linked glycans, O-GalNAc, etc.) organizes glycosyltransferase genes into pathway-specific and non-pathway-specific steps in the biosynthesis of different glycoconfigurations and illustrates the potential redundancy of individual biosynthesis steps provided by isozymes and other competing biosynthesis steps. Together, these maps provide predictions of structural glycan features influenced by the presence or absence of individual genes, and whether global or differential nuances are expected. Nevertheless, glycosylation pathway mapping needs to be continually refined to add insight into the non-redundant and competing functions of isozymes, which will require further research on isozygous cellular models through combinatorial engineering to assess the results.
Fig.2 Glycosylation pathway of O-GalNAc. (Narimatsu, et al., 2019)0
CD BioGlyco has established an advanced and robust glycan display platform. Our R&D team provides high-quality glycan-engineered cell constructs to clients all over the world. Please feel free to contact us for more details about our services.
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