期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 276, 期 45, 页码 42311-42321出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M101441200
关键词
-
资金
- NHLBI NIH HHS [5-P01-HL41484, 5-R01-HL-58479] Funding Source: Medline
- NIGMS NIH HHS [GM-50573] Funding Source: Medline
Using recombinant retroviral transduction, we have introduced the heparin/heparan sulfate (HS) 3-O-sulfotransferase I (3-OST-1) gene into Chinese hamster ovary (CHO) cells. Expression of 3-OST-1 confers upon CHO cells the ability to produce anticoagulantly active HS (HSact). To understand how 6-OST and other proteins regulate HSact biosynthesis, a CHO cell clone with three copies of 3-OST-1 was chemically mutagenized. Resulting mutants that make HS but are defective in generating HSact were single-cell-cloned. One cell mutant makes fewer 6-O-sulfated residues. Modification of HS chains from the mutant with pure 6-OST-1 and 3'-phosphoadenosine 5'-phosphosulfate increased HSact from 7% to 51%. Transfection of this mutant with 6-OST-1 created a CHO cell line that makes HS, 50% of which is HSact. We discovered in this study that M 6-OST-1 is a limiting enzyme in the HSact biosynthetic pathway in vivo when the limiting nature of 3-OST-1 is removed; (ii) HS chains from the mutant cells serve as an excellent substrate for demonstrating that 6-OST-1 is the limiting factor for Hs(act) generation in vitro; (iii) in contradiction to the literature, 6-OST-1 can add 6-O-sulfate to GlcNAc residues, especially the critical 6-O-sulfate in the antithrombin binding motif; (iv) both 3-O- and 6-O-sulfation can be the final step in HSact biosynthesis in contrast to prior publications that concluded 3-O-sulfation is the final step in HSact biosynthesis; (v), in the presence of HS interacting protein peptide, 3-O-sulfate-containing sugars can be degraded into disaccharides by heparitinase digestion as demonstrated by capillary high performance liquid chromatography coupled with mass spectrometry.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据