Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine. PLoS One 2012;7(3):e32762
Date
03/10/2012Pubmed ID
22403705Pubmed Central ID
PMC3293841DOI
10.1371/journal.pone.0032762Scopus ID
2-s2.0-84863231967 (requires institutional sign-in at Scopus site) 23 CitationsAbstract
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.
Author List
Hoffmann BR, Liu Y, Mosher DFMESH terms used to index this publication - Major topics in bold
AcetylglucosamineAmino Acid Sequence
Animals
Consensus Sequence
Drosophila melanogaster
Epidermal Growth Factor
Extracellular Space
Humans
Molecular Sequence Data
Oxygen
Sequence Analysis, DNA
Thrombospondin 1