6-O-sulfotransferase-1 represents a critical enzyme in the anticoagulant heparan sulfate biosynthetic pathway.
Using recombinant retroviral transduction, we have introduced the heparin/heparan sulfate (HS) 3-O-sulfotransferase 1 (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 (HS(act)). To understand how 6-OST and other proteins regulate HS(act) 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 HS(act) 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 HS(act) from 7% to 51%. Transfection of this mutant with 6-OST-1 created a CHO cell line that makes HS, 50% of which is HS(act). We discovered in this study that (i) 6-OST-1 is a limiting enzyme in the HS(act) 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 HS(act) biosynthesis in contrast to prior publications that concluded 3-O-sulfation is the final step in HS(act) 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.
Zhang
L
L
Department of Biology, Division of Bioengineering, Environmental Health Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Beeler
D L
DL
Lawrence
R
R
Lech
M
M
Liu
J
J
Davis
J C
JC
Shriver
Z
Z
Sasisekharan
R
R
Rosenberg
R D
RD
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis; D015238:Sulfotransferases / Q000235:genetics / Q000502:physiology*
D000925:Anticoagulants; D006497:Heparan Sulfate; D012333:RNA, Messenger; D015238:Sulfotransferases; C485757:HS6ST2 protein, human; C404189:heparan sulfate 6-O-sulfotransferase