It remains a significant challenge to predict a priori what sites in a protein can/will become glycosylated and what effect glycan installation at a particular site will have on protein structure and function. Shotgun scanning mutagenesis combines the concepts of alanine scanning mutagenesis and binomial mutagenesis with phage display technology, allowing an extremely rapid method for analyzing the effect of many side chains. At present, there is no analogous shotgun scanning technique for rapid, high-throughput determination of many glycan sites in a single experiment. To address this technology gap, we have developed an experimental procedure termed shotgun scanning glycomutagenesis (SSGM) for comprehensively identifying the sites/structural motifs within a target protein that can/cannot tolerate glycan installation. SSGM uses combinatorial protein libraries in which glycosylation “sequon walking” is used to introduce an acceptor site at every possible position along the protein backbone. The library of sequon variants is interrogated for the presence/absence of glycosylation using a high-throughput screen, such as our previously described glycoSNAP (glycosylation of secreted N-linked acceptor proteins) technology. As proof-of-concept, we used SSGM to investigate the permissiveness to glycosylation of the Im7 immunity protein that has been extensively studied including with respect to limited site-specific glycosylation. Following library construction and screening, a surprisingly large number of sites along Im7 were found to be glycosylated, with efficiencies ranging from 30-100%. The binding activities of all the glycovariants with or without glycan were assessed. The melting temperature of the selected variants was interrogated. We anticipate that SSGM will become a useful new tool for deep exploration of glycoprotein structure-activity relationships (glycoSARS) as well as for developing glycoengineered protein variants with new or improved functions.

Rapid mapping of glycoprotein structure-activity relationships by glycomutagenesis

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.