LAUSR

Significance While it is well established that heavily O-glycosylated mucins are essential for normal gastrointestinal function, it is not known how polypeptide N-acetylgalactosaminyl transferase (GalNAc-T12)–specific O-glycans may be contributing. Here we use structural, computational, and biochemical methods to show how the GALNT12 mutations present in patients with colorectal cancer disrupt the enzymatic function of GalNAc-T12. This information will help identify specific biological substrates of GalNAc-T12 and serve to inform our understanding of their functions, including their potential contributions to cancer initiation and progression. Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.