ENZYMATIC SULFATION OF GALACTOSE RESIDUE OF KERATAN SULFATE BY CHONDROITIN 6-SULFOTRANSFERASE

We have previously found that the purified chondroitin 6-sulfotransfer ase (C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosph osulfate (PAPS) to position 6 of N-acetylgalactosamine in chondroitin, catalyzed the sulfation of keratan sulfate, and that both the C6ST ac tivity and the keratan sulfate sulfotransferase (KSST) activity were e xpressed in COS-7 cells when C6ST cDNA was transfected, In this report we describe some properties of the KSST activity contained in the pur ified C6ST, and characterize the sulfated products formed from keratan sulfate and partially desulfated keratan sulfate, Optimal pH, require ment for cationic activators, and K-m value for PAPS of the KSST activ ity were very similar to those of the C6ST activity. S-35-Labeled glyc osaminoglycans formed from keratan sulfate and partially desulfated ke ratan sulfate were N-deacetylated by treatment with hydrazine/hydrazin e sulfate and then cleaved with HNO2 at pH 4, and the resulting produc ts were reduced with (NaBH4)-H-3. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred sulfate to position 6 of galac tose residue which was glycosidically linked to N-acetylglucosamine 6- sulfate residue or to N-acetylglucosamine residue, Northern blot analy sis using poly (A)(+) RNA from 12-d-old chick embryos indicated that t he message of C6ST was expressed not only in the cartilage but also in the cornea in which keratan sulfate is actively synthesized.