title: Bioengineered Chinese hamster ovary cells with Golgi-targeted 3-O-sulfotransferase-1 biosynthesize heparan sulfate with an antithrombin-binding site.
abstract: HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.
authors.affiliation: From the Departments of Biology.
authors.forename: Payel
authors.initials: P
authors.lastname: Datta
authors.forename: Guoyun
authors.initials: G
authors.lastname: Li
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Xue
authors.initials: X
authors.lastname: Zhao
authors.forename: Jong Youn
authors.initials: JY
authors.lastname: Baik
authors.forename: Trent R
authors.initials: TR
authors.lastname: Gemmill
authors.forename: Susan T
authors.initials: ST
authors.lastname: Sharfstein
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006056:Golgi Apparatus / Q000201:enzymology* / Q000235:genetics; D006493:Heparin / Q000096:biosynthesis* / Q000235:genetics; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D060847:Metabolic Engineering; D051379:Mice; D015238:Sulfotransferases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000581:Amidohydrolases; D006493:Heparin; D006497:Heparan Sulfate; D015238:Sulfotransferases; C113066:NDST2 protein, human; C491855:Ndst2 protein, mouse; C021350:heparitin sulfotransferase
keywords: Antithrombin; Golgi; Heparan Sulfate; Heparin; Metabolic Engineering; Sulfotransferase