GlycoLab: Functional Glycobiology

Bacterial surfaces are decorated with distinct carbohydrate structures that may substantially differ among species and strains. These structures can be recognized by a variety of glycan-binding proteins, playing an important role in the bacteria cross-talk with the host and invading bacteriophages, and also in the formation of bacterial microcolonies and biofilms. In recent years, different microarray approaches for exploring bacterial surface glycans and their recognition by proteins have been developed. A main advantage of the microarray format is the inherent miniaturization of the method, which allows sensitive and high-throughput analyses with very small amounts of sample. Antibody and lectin microarrays have been used for examining bacterial glycosignatures, enabling bacteria identification and differentiation among strains. In addition, microarrays incorporating bacterial carbohydrate structures have served to evaluate their recognition by diverse host/phage/bacterial glycan-binding proteins, such as lectins, effectors of the immune system, or bacterial and phagic cell wall lysins, and to identify antigenic determinants for vaccine development. The list of samples printed in the arrays includes polysaccharides, lipopoly/lipooligosaccharides, (lipo)teichoic acids, and peptidoglycans, as well as sequence-defined oligosaccharide fragments. Moreover, microarrays of cell wall fragments and entire bacterial cells have been developed, which also allow to study bacterial glycosylation patterns. In this review, examples of the different microarray platforms and applications are presented with a view to give the current state-of-the-art and future prospects in this field.

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on O- and N-glycans of various glycoproteins and on glycolipids. It is overexpressed in the nervous system and plays crucial roles in nerve regeneration, synaptic plasticity, and neuronal diseases. However, the investigation of functional roles of HNK-1 in a more complex glycan context at the molecular level remains a big challenge due to lack of access to related structurally well-defined complex glycans. Herein, we describe a highly efficient chemoenzymatic approach for the first collective synthesis of HNK-1-bearing O-mannose glycans with different branching patterns, and for their nonsulfated counterparts. The successful strategy relies on both chemical glycosylation of a trisaccharide lactone donor for the introduction of sulfated HNK-1 branch and substrate promiscuities of bacterial glycosyltransferases that can tolerate sulfated substrates for enzymatic diversification. Glycan microarray analysis with the resulting complex synthetic glycans demonstrated their recognition by two HNK-1-specific antibodies including anti-HNK-1/N-CAM (CD57) and Cat-315, which provided further evidence for the recognition epitopes of these antibodies and the essential roles of the sulfate group for HNK-1 glycan-antibody recognition.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors that plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-$\beta$. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally-defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-$\beta$ secretion following co-culture of Siglec-15-expressing monocytic cells lines with tumor cells expressing sTn, or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.