For the subcellular localization experiments, HeLa (FUT2) cells were seeded onto glass #1.5 coverslips precoated with 100 μg/ml poly-D-lysine and transfected using jetOPTIMUS reagent following Polyplus’s instructions. 0.175 μg of each myc and HA construct and 0.15 μg of pGTS-BFP were introduced per sample. After 16 h, cells were fixed with paraformaldehyde 4%. COS1(B3GALNT1) cells or HeLa (FUT2) cells were seeded on 12-well plates for cytometry experiments. The day after reaching around 80–90% confluence, cells were using jetOPTIMUS following Polyplus’s instructions. 0.9 μg of sample DNA and 0.1 μg of pmCherry-N1 or pEGFP-N1 were transfected per well. The cells were analyzed 16 h post-transfection.

Some stem region mutants of human blood group A transferase (hAT) possess Forssman synthase (FS) activity, but very little is known about the mechanisms responsible for this enzymatic crosstalk. We performed confocal microscopy and image analysis to determine whether different intra-Golgi localization was accountable for this acquired activity. We also performed structural modeling and mutational and normal mode analyses. We introduced new mutations in the stem region and tested its FS and AT activities. No differences in subcellular localization were found between hAT and FS-positive mutants. AlphaFold models of hAT and mFS (mouse Forssman synthase) showed that the hAT stem region has a tether-like stem region, while in mFS, it encircles its catalytic domain. In silico analysis of FS-positive mutants indicated that stem region mutations induced structural changes, decreasing interatomic interactions and mobility of hAT that correlated with FS activity. Several additional mutations introduced in that region also bestowed FS activity without altering the AT activity: hAT 37-55 aa substitution by mFS 34-52, 37-55 aa deletion, and missense mutations: S46P, Q278Y, and Q286M. Stem region structure, mobility, and interactions are crucial for hAT specificity. Moreover, stem region mutations can lead to heterologous Forssman activity without changes in the catalytic machinery.