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. 2022 Jan 30;298(3):101666. doi: 10.1016/j.jbc.2022.101666

Figure 4.

Figure 4

The in vitro activity of soluble GnT-V WT and ΔN toward various substrates measured by the UDP-Glo assay.A, treatment of the glycoproteins used in this assay with sialidase and galactosidase. The treated proteins were stained with CBB, SSA, or RCA lectin. B, schematic diagram of substrate preparation by glycosidase treatments for the UDP-Glo assay (left). The UDP-Glo assay was performed using purified soluble GnT-V WT and glycosidase-treated or -untreated substrates (SOD3 or SGP) (right). C, comparison of the in vitro activity of GnT-V WT and GnT-VΔN toward various types of substrates using the UDP-Glo assay. The relative activity toward each substrate was evaluated by setting the activity of GnT-V WT toward GnGnbi-PA as 1 (n = 3, mean ± S.D. ∗p < 0.05). The p value was calculated by 2-way ANOVA Dunnett’s test. D, base peak chromatogram (BPC) from LC-ESI-MS analysis of N-glycans from transferrin which had been incubated with GnT-V WT, ΔN, or vehicle. Extracted ion chromatograms (EICs) of the substrate biantennary glycan and the product triantennary glycan are also shown. E, the ratios of signal intensity of the product glycan to that of total (substrate + product) glycans in (D) are shown. GnT-V, N-acetylglucosaminyltransferase-V; PA, 2-aminopyridine; RCA, Ricinus communis agglutinin; SGP, chicken egg sialylglycopeptide; SOD3, superoxide dismutase 3; SSA, Sambucus sieboldiana agglutinin.