Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase.
We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.
Ohtake
Shiori
S
Department of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan.
Kimata
Koji
K
Habuchi
Osami
O
D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013431:Sulfates; D002809:Chondroitin Sulfates; D015238:Sulfotransferases; C118596:UST protein, human; D002808:Chondroitin Lyases