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A peptide panel investigation reveals the acceptor specificity of O ‐GlcNAc transferase
Author(s) -
Liu Xiaoyan,
Li Ling,
Wang Yuqiu,
Yan Hui,
Ma Xiaofeng,
Wang Peng George,
Zhang Lianwen
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.13-246850
Subject(s) - amino acid , peptide , mutagenesis , peptide sequence , chemistry , stereochemistry , sequence (biology) , random coil , transferase , amino acid residue , western blot , biochemistry , enzyme , mutant , protein secondary structure , gene
O ‐linked β‐ N ‐acetylglucosaminylation ( O ‐GlcNAcylation) is widely distributed on nucleocytoplasmic proteins and participates in various physiological processes. But O ‐GlcNAc status on numerous proteins remains unknown. To better understand this modification, computational analysis combined with experimental study was performed in this work. Structural analysis of many O ‐GlcNAcylation sites indicated that the modification occurred predominantly in a random coil region. Frequency analysis on many O ‐GlcNAcylated peptides revealed a signature sequence, PPV S/T SATT, around the modification site (underlined, position 0). Based on the sequence, a peptide panel was designed to investigate key positions affecting O ‐GlcNAcylation of peptides and their amino acid preference. It was indicated that 3 positions (–2, –1, and +2) had an important role for this modification, where the presence of uncharged amino acids with small side chains could confer high reactivity. The amino acid preference at key positions was further investigated on bovine crystalline α via site‐directed mutagenesis. The preferred amino acids were Pro > Ala > Gly at position – 2, Ala > Thr > Val > Lys > Pro at position –1, and Ala > Gly > Arg > Glu at position +2. Altogether, these findings suggested that a substrate (peptide or protein) with Pro, Ala at position –2, and/or Val, Ala, Thr, Ser at position –1, and/or Ala, Ser, Pro, Thr, Gly at position +2 would have more chances for O ‐GlcNAcylation. To test the rule, 2 O ‐GlcNAcylation sites on sOGT (S52 and T449) were predicted and confirmed by Western blot. The present work systematically investigated the sequence signature for O ‐GlcNAcylation. The result will contribute to predicting the O ‐GlcNAc status of a protein and further functional studies.—Liu, X., Li, L., Wang, Y., Yan, H., Ma, X., Wang, P. G., Zhang, L. A peptide panel investigation reveals the acceptor specificity of O ‐GlcNAc transferase. FASEB J. 28, 3362–3372 (2014). www.fasebj.org