Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

Area of Science:

• Glycobiology
• Biochemistry
• Cell Biology

Background:

• Heparan sulfate (HS) is a critical polysaccharide involved in numerous biological processes.
• T/Tn antigens are recognized as universal carbohydrate cancer markers.
• Accurate imaging of these glycans is essential for understanding their roles.

Purpose of the Study:

• To develop and validate specific imaging methods for heparan sulfate (HS) and T/Tn antigens.
• To investigate the architectural features of the extracellular matrix (ECM) in HUVEC cells using HS staining.
• To explore the kinetics of O-glycan synthesis, specifically the role of O-GalNAcylation.

Main Methods:

• Utilized a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVECs) for imaging.
• Employed comparative imaging of various glycans to demonstrate staining specificities.
• Validated specificity through glycan removal (loss of signal) and installation (gain of signal).

Main Results:

• HS staining revealed previously undescribed architectural features of the HUVEC ECM.
• T/Tn antigen staining indicated that O-GalNAcylation is a rate-limiting step in O-glycan synthesis.
• Specific imaging was achieved for HS, T/Tn antigens, O-GlcNAc, lactosaminyl glycans, and hyaluronan.

Conclusions:

• Developed highly specific imaging approaches for HS and T/Tn antigens.
• The findings facilitate the detection and functional characterization of these important glycans.
• The study provides new insights into ECM structure and glycan synthesis pathways.