Presentation, presentation, presentation! Molecular-level insight into linker effects on glycan array screening data

Abstract

Changes in cell-surface glycan patterns are markers of the presence of many different disease and cancer types, offering a relatively untapped niche for glycan-targeting reagents and therapeutics in diagnosis and treatment. Of paramount importance for the success of any glycan-targeting reagent is the ability to specifically recognize the target among the plethora of different glycans that exist in the human body. The preeminent technique for defining specificity is glycan array screening, in which a glycan-binding protein (GBP) can be simultaneously screened against multiple glycans. Glycan array screening has provided unparalleled insight into GBP specificity, but data interpretation suffers from difficulties in identifying false-negative binding arising from altered glycan presentation, associated with the linker used to conjugate the glycan to the surface. In this work, we model the structure and dynamics of the linkers employed in the glycan arrays developed by the Consortium for Functional Glycomics. The modeling takes into account the physical presence and surface polarity of the array, and provides a structure-based rationalization of false-negative results arising from the so-called “linker effect.” The results also serve as a guide for interpreting glycan array screening data in a biological context; in particular, we show that attempts to employ natural amino acids as linkers may be prone to unexpected artifacts compromising glycan recognition.

The Tetrameric Plant Lectin BanLec Neutralizes HIV through Bidentate Binding to Specific Viral Glycans

“…A model for bound trimannose oligosaccharide (Manα1-2Manα1-3Manα) was built by grafting (Grant et al, 2014; Tessier et al, 2013) the required residues onto the co-crystal structure of Manα bound to BanLec (PDBID: 2BMZ).…”

Induction of Antibodies Directed Against Branched Core O‐Mannosyl Glycopeptides—Selectivity Complimentary to the ConA Lectin

“…The glycopeptide microarray was also employed to evaluate the binding specificity of ConA to the assorted O -mannosyl glycan structures. Computational carbohydrate grafting [23] was performed on the O -mannosyl glycan–ConA crystal structure complexes (taken from PDB ID 1TEI) to gain a structural understanding of the observed specificities.…”

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

“…The covalent immobilization procedures discussed above are thought to lead to a distribution of glycans with different orientations in space and a high degree of flexibility, a situation preferred for screening purposes. Still, effects of the linker between the glycans and slide matrix will play a role, as shown for the CFG array (NHS chemistry), where even minor differences in linker design severely affected glycan recognition by an mAb both in silico and experimentally (49). Furthermore, typical screening arrays are produced at high surface glycan density (50), and although this parameter can be influenced by glycan printing Neoglycoprotein: glycoprotein established by chemically attaching glycans to a nonglycosylated protein concentration, a more systematic approach to investigating density influence on GBP binding was developed using arrays printed with neoglycoproteins that varied in the number of attached glycans (51).…”