Genetically-encoded fragment-based discovery (GE-FBD) of glycopeptide ligands with differential selectivity for antibodies related to mycobacterial infections

Abstract

Accurate identification of tuberculosis (TB), caused by Mycobacterium tuberculosis, is important for global disease management. Point-of-care serological tests may improve TB diagnosis; however, specificities of available serodiagnostics are sub-optimal. We employed genetically encoded fragment-based discovery (GE-FBD) to select ligands for antibodies directed against the mycobacterial cell wall component lipoarabinomannan (LAM), a potent antigen. GE-FBD employed a phage displayed library of 10 heptapeptides, chemically modified with an arabinofuranosyl hexasaccharide fragment of LAM (Ara), and the anti-LAM antibody CS-35 as a bait. The selection gave rise to glycopeptides with an enhanced affinity and selectivity for CS-35 but not for 906.4321 antibody, both of which bind to Ara with a comparable affinity. Multivalent assays incorporating the discovered ligands Ara-ANSSFAP, Ara-DAHATLR and Ara-TTYVVNP exhibited up to 19-fold discrimination between CS-35 and 906.4321. The use of the Ara antigen alone failed to distinguish these antibodies. Thus, GE-FBD gives rise to ligands that differentiate monoclonal antibodies with enhanced specificity. This technology could facilitate the development of effective point-of-care serological tests for mycobacterial and other infections.

Selection of galectin‐3 ligands derived from genetically encoded glycopeptide libraries

“…The above differential enrichment (DE) analysis also compared the screen of glycosylated and unmodified libraries and discarded sequences that were equally enriched with and without glycosylation (Figure D). The latter filtering strategy has previously shown to be effective in discovery of peptides that bind to GBP in synergy with glycan . Intersect of the 3 DE analyses yielded 1061 glycopeptides that satisfied all criteria (Figure E).…”

“…We previously demonstrated that genetically encoded fragment‐based discovery (GE‐FBD) in which the glycan fragment is covalently linked to library of ten peptide fragments can minimize challenges of classical genetically encoded peptide libraries, high‐throughput screens, and fragment‐based discovery alone. In several reports, GE‐FBD applied to glycan‐binding proteins yielded peptide–glycan combinations 10‐50 times more potent than the original glycan fragment . In GE‐FBD, the carbohydrate fragment directs the interactions of peptide and target to the CRD and the screening process could identify peptides that provide synergistic binding contributions that enhance overall binding affinity.…”

“…A few questions however remain open about GE‐FBD: (1) what is the upper and lower limit of affinity for the initial fragment? Previous GE‐FBD‐like approaches employed fragments with affinities of 10‐100 fM, 1.2 nM 9 , 1 μM, 60 μM, 150 μM, and 1 mM 4 . (2) Can GE‐FBD work effectively when it employs a mixture of glycan fragments?…”

“…(2) Can GE‐FBD work effectively when it employs a mixture of glycan fragments? With one exception, all GE‐FBD approaches applied to date used only 1 glycan or one type of fragment . To address these fundamental questions, we employed GE‐FBD to conduct discovery of glycopeptide ligands for GBPs called galectins starting from a mixture of fragments that have low (10 mM) or no detectable affinity for a galectin protein.…”

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

“…[37] These reactions need to be extremely selective to be suitable for phage or mRNA displayed libraries without damaging the requisite phage particle or nucleic acid tag. [36,38] Of particular note is that even alow-affinity glycan can be used to target the site of binding,t hus increasing the chance of discovering ag lycopeptide with the desired biological activity. [36,38] Of particular note is that even alow-affinity glycan can be used to target the site of binding,t hus increasing the chance of discovering ag lycopeptide with the desired biological activity.…”