{"doi":"10.17615/n83m-cm58","title":"Using Engineered 2- O -Sulfotransferase to Determine the Activity of Heparan Sulfate C 5 -epimerase and Its Mutants","abstract":"Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C5-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically 3H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C5-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C5-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C5-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C5-epimerase, proving that this two-enzyme coupling system can be used to assay for C5-epimerase. The method was further used to determine the activities of various C5-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C5-epimerase and facilitate the structural and functional analysis of C5-epimerase.","journal":"UNC Libraries","year":2020,"id":135447,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9638,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":592197,"name":"Kai Li","orcid":"0000-0003-2963-005X","position":1,"is_corresponding":false},{"id":288986,"name":"Jian Liu","orcid":"0000-0001-8552-1400","position":2,"is_corresponding":false},{"id":592933,"name":"Heather N. Bethea","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:16:31.787520Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}