Engineering glycosyltransferases into glycan binding proteins using a novel mammalian surface display platform

Traditional lectins that bind cell-surface carbohydrate epitopes exhibit broad binding specificity. We tested the hypothesis that more specific glycan-binding proteins may be developed by protein engineering of mammalian glycosyltransferases. Using rational design, we observed that introducing an H302A mutation into porcine ST3Gal1, while abolishing enzymatic activity, resulted in a lectin displaying specific binding to sialylated core-2 O -linked glycans (i.e. Neu5Acα2-3Galβ1–3[GlcNAc(β1–6)]GalNAcα epitope). To expand on this, we developed a novel mammalian cell-surface display platform to screen for additional variants. One ST3Gal1 mutant (called ‘sCore2’) containing three mutations H302A/A312I/F313S exhibited improved binding properties. Spectral flow cytometry analysis with sCore2 shows that individual blood cell types exhibit unique O -glycosylation profiles. Tissue microarray analysis also reveals unique cell/tissue specific staining patterns of sCore2. Overall, glycosyltransferases can be engineered to yield specific glycan binding proteins, suggesting that a similar approach may be extended to other glycoenzymes also.