Figure 2

Relative mobilities of various Cy5-labeled glycans and their enzymatic synthesis. All glycans were enzymatically synthesized starting from the glycan A2 (known as G0 in IgG glycan notation). The Glycan ladders were composed by combining some of the labeled glycans. With the exception of the enzymatic reactions of FUT8, MGAT3 and MGAT5, all other enzymatic reactions resulted in one or two intermediates as there are two or more branches in each glycan that allow enzymatic modification (see Supplemental Fig. 1 for intermediates). The labeled glycans were separated on a 17% gel and visualized with a fluorescent imager. (A) Relative mobilities of glycans that were labeled at the core-fucose. The enzymes used for generating these glycans are listed on the top of the image. (B) Relative mobility change on FA2 by addition of a bisecting GlcNAc introduced by MGAT3 (MT3) versus a β1-6GlcNAc introduced by MGAT5 (MT5). MGAT3 and MGAT5 were introduced in different orders. The 2nd enzyme was introduced 30 min after the 1st enzyme. FA3B containing both a bisecting and a β1-6 GlcNAc can only be observed when MGAT5 was introduced first. (C) Relative mobility change on A2G2S(6)1 by addition of a bisecting GlcNAc versus a core-fucose. The glycans were generated and labeled starting from A2 with the enzymes indicated at the top of the gel in the specified order. MT3, MGAT3; FT8, FUT8; B41, B4GalT1; ST61, ST6Gal1. (D) Schemes for enzymatic generation of the labeled glycans in (A) and (B). Labeling was through FUT8 and GDP-Cy5-Fuc (GDP-f’). (E) Schemes for enzymatic generation of the labeled glycans in (C). Labeling was through ST6Gal1 and CMP-Cy5-Neu5Ac (CMP-S’). Two Cy5-Neu5Ac residues can be introduced to each of the glycans, but only glycans with one Cy5-Neu5Ac were displayed in (B), (C) and (E).