A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase.
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, PPVS/TSATT, 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.
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Xiaoyan
X
Liu
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Ling
L
Li
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Yuqiu
Y
Wang
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Hui
H
Yan
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Xiaofeng
X
Ma
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Peng George
PG
Wang
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China lianwen@nankai.edu.cn.
Lianwen
L
Zhang
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D030562:Databases, Protein; D005813:Genes, Synthetic; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D038201:alpha-Crystallins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
OGT; amino acid preference