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J Biol Chem, Vol. 274, Issue 53, 38155-38162, December 31, 1999

Heparan Sulfate D-Glucosaminyl 3-O-Sulfotransferase-3A Sulfates N-Unsubstituted Glucosamine Residues^*

Jian Liu^§, Zach Shriver^¶, Peter Blaiklock, Keiichi Yoshida, Ram Sasisekharan^¶, and Robert D. Rosenberg^§**

From the  Department of Biology and ^¶ Division of Bioengineering and Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the ^§ Molecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts 02215, and the  Tokyo Research Institute of Seikagaku Corporation, Higashiyamato-shi, Tokyo 207, Japan

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-³⁵S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of UA2S-GlcNS-IdoUA2S-[³⁵S]GlcNH₂3S and UA2S-GlcNS-IdoUA2S-[3-³⁵S]GlcNH₂3S6S. The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N-unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3-OST-3A-modified heparan sulfate.

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^* This work was supported by National Institutes of Health Grants 5.P01.HL41484 (to R. D. R.) and R01.GM57073 (to R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence and reprint requests should be addressed: 68-480, Massachusetts Institute of Technology, 31 Ames St., Cambridge, MA 02139. Tel.: 617-253-8803; Fax: 617-258-6553.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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