GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins

In
2011, a new type of protein O-glycosylation
was discovered in which N-acetylgalactosamine is
attached to the side chain of tyrosine (GalNAc-Tyr). While present
on dozens of proteins, the biological roles of GalNAc-Tyr are unknown.
To gain insight into this new type of modification, we synthesized
a group of GalNAc-Tyr glycopeptides, constructed microarrays, and
evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding
proteins. Through a series of >150 microarray experiments, we assessed
binding properties of a variety of plant lectins, monoclonal antibodies,
and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly
to GalNAc-Tyr, and several Tn binding antibodies and blood group A
antibodies were found to cross-react with GalNAc-Tyr. Thus, detection
of GalNAc-Tyr modified proteins is an important consideration when
analyzing results from these reagents. Additionally, we evaluated
potential recognition by two mammalian lectins, human (hMGL) and murine
(mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2
bound tightly to GalNAc-Tyr determinants. The apparent Kd values (∼1–40 nM) were on par with some
of the best known ligands for MGL, such as the Tn antigen. hMGL also
bound the natural beta-amyloid peptide containing a GalNAc-Tyr epitope.
STD NMR experiments provided structural insights into the molecular
basis of recognition. Finally, GalNAc-Tyr was selectively captured
by mMGL-2 positive dendritic cells. These results provide the first
evidence that GalNAc-Tyr modified proteins and/or peptides may be
ligands for hMGL and mMGL-2 and offer unique structures for the design
of MGL targeting agents.