Himori, K.; Zhang, B.; Matsui, Y.

Enzyme-centered interpretation of glycomic changes requires testing whether glycans associated with particular reactions change collectively. Motif analyses and reaction databases do not directly provide enzyme-specific sets of measured glycans, and structural ambiguity or overlapping reactions can give different enzymes identical sets. A framework is needed to construct these sets and define distinguishable test hypotheses. GlyEnzEA constructs sets by matching reaction rules to candidate glycan topologies, groups enzymes sharing identical sets, and tests each set once. In an Mgat3-knockout mouse glycome, 48 enzymes yielded 16 sets; only the MGAT3 set showed significant negative enrichment after correction (Storey q=0.033). Across seven HEK293 perturbations, target sets were significant in two conditions: MGAT1 and class-I mannosidase knockouts. GlyEnzEA supports enzyme-centered interpretation at the resolution of distinguishable enzymes or enzyme groups.