Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A sulfates N-unsubstituted glucosamine residues.
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-(35)S-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 DeltaUA2S-GlcNS-IdoUA2S-[(35)S]GlcNH(2)3S and DeltaUA2S-GlcNS-IdoUA2S-[3-(35)S]GlcNH(2)3S6S . 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.
Liu
J
J
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Shriver
Z
Z
Blaiklock
P
P
Yoshida
K
K
Sasisekharan
R
R
Rosenberg
R D
RD
D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013431:Sulfates / Q000737:chemistry; D015238:Sulfotransferases / Q000737:chemistry* / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D013431:Sulfates; D015238:Sulfotransferases; C102180:heparan sulfate D-glucosaminyl 3-O-sulfotransferase; D005944:Glucosamine