MOLECULAR-CLONING AND CHARACTERIZATION OF HUMAN KERATAN SULFATE GAL-6-SULFOTRANSFERASE

We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA f rom chick embryo chondrocytes. C6ST catalyzes sulfation of chondroitin , keratan sulfate, and sialyl N-acetyllactosamine oligosaccharides. In this study, we report the cloning and characterization of a novel sul fotransferase that catalyzes sulfation of keratan sulfate, This new su lfotransferase cDNA clone was obtained from a human fetal brain librar y by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmem brane protein composed of 411 amino acid residues, When the cDNA was i ntroduced into a eukaryotic expression vector and transfected in COS-7 cells, keratan sulfate sulfotransferase activity was overexpressed, b ut C6ST activity was not increased over that of the control. Structura l analysis of S-35-labeled glycosaminoglycan, which was formed from ke ratan sulfate by the reaction with S-35-labeled 3'-phosphoadenosine 5' -phosphosulfate and the recombinant sulfotransferase, showed that kera tan sulfate was sulfated at position 6 of Gal residues, On the basis o f the acceptor substrate specificity, we propose keratan sulfate Gal-6 -sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotra nsferase. KSGal6ST was assigned to chromosome 11p11.1-11.2 by fluoresc ence in situ hybridization. Among various human adult tissues, a 2.8-k ilobase message of KSGal6ST was expressed mainly in the brain. When po ly(A)(+) RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with a bout 2.8 kilobases was detected. These observations suggest that KSGal 6ST may participate in the biosynthesis of keratan sulfate in the brai n and cornea.