title: Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine.
abstract: 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.
authors.affiliation: Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, United States of America.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Hoffmann
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Deane F
authors.initials: DF
authors.lastname: Mosher
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D004331:Drosophila melanogaster; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010100:Oxygen; D017422:Sequence Analysis, DNA; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D019700:Thrombospondin 1; D004815:Epidermal Growth Factor; D010100:Oxygen; D000117:Acetylglucosamine