Single chain antibodies that recognize the N-glycosylation site

Abstract

We aimed to identify antibodies that can recognize the Asn–Xaa–Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn–Cys–Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with K d values of the order of 10 −6 M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn–Pro–Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.