Introduction of extended LEC14‐type branching into core‐fucosylated biantennary N‐glycan

Abstract

A series of enzymatic substitutions modifies the basic structure of complex‐type biantennary N‐glycans. Among them, a β1,2‐linked N ‐acetylglucosamine residue is introduced to the central mannose moiety of the core‐fucosylated oligosaccharide by N ‐acetylglucosaminyltransferase VII. This so‐called LEC14 epitope can undergo galactosylation at the β1,2‐linked N ‐acetylglucosamine residue. Guided by the hypothesis that structural modifications in the N‐glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N‐glycan and tested its properties in three different assays. In order to allow comparison to previous results on other types of biantennary N‐glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant. Compared to the core‐fucosylated N‐glycan no significant change in affinity was seen when testing three galactoside‐specific proteins. However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines. Analysis of biodistribution in tumor‐bearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor/blood ratio. Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core‐fucosylated biantennary N‐glycan. The results argue in favor of the concept of viewing substitutions as molecular switches for lectin‐binding affinity. Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging.