FIGURE 5.

Characterization of CcGnt1 (A–C) and its glycan product in vitro (D). A–C, in vitro reaction with variable conditions were performed and the qualitative glycan educt to product conversion was analyzed by MALDI-TOF-MS. Glycans with the mass of 1377 Da (educt) and 1580 Da (product) are indicated schematically above the spectra. A, standard conditions with crude extract from cells transfected with vector control or with CcGnt1, respectively, were compared. B, influence of divalent cations on the enzyme activity. The reaction was performed with either Mn²⁺ (standard), Mg²⁺ or EDTA. C, to determine the transferase specificity, either UDP-GlcNAc or UDP-GalNAc was used as sugar donor. D, product was analyzed in more detail. In vitro reaction product was digested with α-mannosidase, purified, and permethylated. MS/MS spectrum of the resulting glycans is shown with peaks corresponding to characteristic ions. These are labeled and illustrated in the Haworth representation above the spectrum. E, scheme summarizing the findings concerning the structure of the reaction product.