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A 6-O-endosulfatase activity assay based on synthetic Heparan Sulfate oligomers.

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Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the post-synthetic editing of heparan sulfate which regulates many important biological processes. Activity of the Sulf-2 and its substrate specificity remain insufficiently… Click to show full abstract

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the post-synthetic editing of heparan sulfate which regulates many important biological processes. Activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well characterized synthetic substrates for probing of its catalytic activity. We introduce synthetic heparan sulfate oligosaccharides to fill this gap and we use our recombinant Sulf-2 protein to show that a p-nitrophenol labeled synthetic oligosaccharide allows reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange HPLC chromatography and quantified by UV-absorbance. This simple assay allows detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that N-glycosylation of the Sulf-2 enzyme affects activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Keywords: heparan; heparan sulfate; synthetic heparan; endosulfatase activity; activity

Journal Title: Glycobiology
Year Published: 2023

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