RESUMEN
Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.
Asunto(s)
Glicoproteínas/química , Indoles/química , Proteínas/química , Sitios de Unión , Cristalografía por Rayos X , Dictyostelium , Fetuínas , Citometría de Flujo , Glicoproteínas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/metabolismo , Polisacáridos/química , Proteínas/metabolismoRESUMEN
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.