Modification of EGF-Like Module 1 of Thrombospondin-1, an Animal Extracellular Protein, by O-Linked N-Acetylglucosamine

Brian R Hoffmann,Deane F Mosher,Yuanyuan Liu,David D Roberts

doi:10.1371/journal.pone.0032762

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 20th 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 575CPPGYSGNGIQC586. These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

Highlights

Thrombospondins (TSPs) are large secreted, calcium-binding glycoproteins
When we compared the sequence of Drosophila Notch that is modified to the sequence of TSP-1, we found similarities in the loop between the fifth and sixth cysteines of the first epidermal growth factor (EGF)-like module, i.e., a serine in TSP-1 and a threonine in Notch are embedded in the sequences PYGSGN and PYGTGQ, respectively (Figure 2)
A BLAST search against the human UniProtKB database with the sequence between the fifth and sixth cysteines of the 20th EGF-like module of Drosophila Notch as bait revealed that a number of human extracellular matrix and transmembrane proteins in addition to TSP-1 contained the sequence CXXG(Y/ F)(T/S)GZ2–5C (X typically a Pro or Ala, Z varying from 2–5 residues) between the fifth and sixth cysteines of EGF-like modules (Figure 2)

Summary

Introduction

Thrombospondins (TSPs) are large secreted, calcium-binding glycoproteins. There are 5 TSPs in humans: 2 trimeric Group A TSPs (TSP-1 and TSP-2) and 3 pentameric Group B TSPs (TSP-3, TSP-4, TSP-5) [1]. Group A TSPs are composed of an N-terminal module (N), an oligomerization sequence (o) that is responsible for trimer formation, a von Willebrand Factor C module (C), three properdin-like modules (P123), three epidermal growth factor (EGF)-like modules (E123), a calcium-binding (Ca) wire, and a globular lectin-like C-terminal module (G) (Figure 1) [1]. Group B TSPs lack the C and P123 modules and contain an extra EGF-like module [1]. Group A TSPs are C-mannosylated on the tryptophans of WXXW sequences in the properdin-like modules, as are other proteins with similar sequences [4,5]. Since extracellular EGF-like modules in Drosophila Notch have recently been identified to contain O-linked b-N-acetylglucosamine (O-b-GlcNAc) [9], we set out to identify any possible O-bGlcNAc modification of TSP-1 EGF-like modules. We report a fourth unusual modification of TSP-1: O-linked b-N-acetylglucosamine (O-b-GlcNAc)

Methods

Results

Conclusion