Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine

PLoS One. 2012;7(3):e32762. doi: 10.1371/journal.pone.0032762. Epub 2012 Mar 5.


Thrombospondin-1 (TSP-1) is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-β-N-acetylglucosaminylation. Previously, O-β-N-acetylglucosamine (O-β-GlcNAc) was found on a threonine in the loop between the fifth and sixth cysteines of the 20(th) epidermal growth factor (EGF)-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with β-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da) and xylose (+132 Da) separately from modification with N-acetyl hexosamine (+203 Da). Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence (575)CPPGYSGNGIQC(586). These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylglucosamine / chemistry*
  • Acetylglucosamine / metabolism*
  • Amino Acid Sequence
  • Animals
  • Consensus Sequence
  • Drosophila melanogaster
  • Epidermal Growth Factor / chemistry*
  • Extracellular Space / metabolism*
  • Humans
  • Molecular Sequence Data
  • Oxygen*
  • Sequence Analysis, DNA
  • Thrombospondin 1 / chemistry*
  • Thrombospondin 1 / metabolism*


  • Thrombospondin 1
  • Epidermal Growth Factor
  • Oxygen
  • Acetylglucosamine