Structural Basis for Peptide Substrate Specificities of Glycosyltransferase GalNAc-T2

Sai Pooja Mahajan, Yashes Srinivasan, Jason W. Labonte, Matthew P. Delisa, Jeffrey J. Gray

Research output: Contribution to journalArticlepeer-review


The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide-enzyme conformations obtained from Rosetta Monte Carlo-minimization-based flexible docking. We computationally scanned 19 amino acid residues at positions -1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19 × 19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and nonglycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5%, and a false positive rate of 7.3%. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the -1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the -1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365, and W331, which modulate the pocket size and specific enzyme-peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide binding suboptimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.

Original languageEnglish (US)
Pages (from-to)2977-2991
Number of pages15
JournalACS Catalysis
Issue number5
StatePublished - Mar 5 2021


  • GalNAcT
  • O-linked glycosylation
  • computational specificity prediction
  • enzyme specificity
  • glycosyltransferases

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)


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