title: Bioengineering murine mastocytoma cells to produce anticoagulant heparin.
abstract: Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
authors: Department of Biology.
authors: Leyla
authors: L
authors: Gasimli
authors: Charles A
authors: CA
authors: Glass
authors: Payel
authors: P
authors: Datta
authors: Bo
authors: B
authors: Yang
authors: Guoyun
authors: G
authors: Li
authors: Trent R
authors: TR
authors: Gemmill
authors: Jong Youn
authors: JY
authors: Baik
authors: Susan T
authors: ST
authors: Sharfstein
authors: Jeffrey D
authors: JD
authors: Esko
authors: Robert J
authors: RJ
authors: Linhardt
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D001709:Biotechnology / Q000379:methods*; D002236:Carbohydrate Conformation; D045744:Cell Line, Tumor; D006493:Heparin / Q000096:biosynthesis* / Q000737:chemistry; D034801:Mastocytoma / Q000378:metabolism; D051379:Mice; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D011994:Recombinant Proteins; D006493:Heparin; D015238:Sulfotransferases; C021350:heparitin sulfotransferase
keywords: heparan sulfate; heparin; mastocytoma; proteoglycan; sulfotransferase