Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase.
We have previously found that the purified chondroitin 6-sulfotransferase (C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine in chondroitin, catalyzed the sulfation of keratan sulfate, and that both the C6ST activity and the keratan sulfate sulfotransferase (KSST) activity were expressed in COS-7 cells when C6ST cDNA was transfected. In this report we describe some properties of the KSST activity contained in the purified C6ST, and characterize the sulfated products formed from keratan sulfate and partially desulfated keratan sulfate. Optimal pH, requirement for cationic activators, and Km value for PAPS of the KSST activity were very similar to those of the C6ST activity. 35S-Labeled glycosaminoglycans formed from keratan sulfate and partially desulfated keratan sulfate were N-deacetylated by treatment with hydrazine/hydrazine sulfate and then cleaved with HNO2 at pH 4, and the resulting products were reduced with NaB3H4. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred sulfate to position 6 of galactose residue which was glycosidically linked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamine residue. Northern blot analysis using poly (A)+ RNA from 12-d-old chick embryos indicated that the message of C6ST was expressed not only in the cartilage but also in the cornea in which keratan sulfate is actively synthesized.
Habuchi
O
O
Department of Life Science, Aichi University of Education, Japan.
Hirahara
Y
Y
Uchimura
K
K
Fukuta
M
M
D000818:Animals; D015152:Blotting, Northern; D001894:Borohydrides; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D004590:Electrophoresis, Paper; D005690:Galactose / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007632:Keratan Sulfate / Q000378:metabolism*; D008969:Molecular Sequence Data; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D001894:Borohydrides; D005690:Galactose; D006025:Glycosaminoglycans; D007632:Keratan Sulfate; D013431:Sulfates; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C025364:sodium borohydride