Format

Send to

Choose Destination
Biochem J. 2018 Aug 14;475(15):2417-2433. doi: 10.1042/BCJ20180265.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Author information

1
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, L69 7ZB Liverpool, U.K.
2
Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, L69 7ZB Liverpool, U.K.
3
Laboratory CRRET CNRS 9215, Université Paris-Est, CRRET (EA 4397/ERL CNRS 9215), UPEC, F-94010 Créteil, France.
4
Glycan Therapeutics, 617 Hutton Street, Raleigh, NC 27606, U.S.A.
5
UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A.
6
Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A.
7
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A.
8
Department of Chemistry, University of Liverpool, L69 7ZD Liverpool, U.K.
9
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, L69 7ZB Liverpool, U.K. patrick.eyers@liverpool.ac.uk.

Abstract

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

KEYWORDS:

heparan sulfate; heparin; inhibitor; kinase; sulfotransferase

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center