Molecular cloning and characterization of human keratan sulfate Gal-6-sulfotransferase.
We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from 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 sulfotransferase that catalyzes sulfation of keratan sulfate. This new sulfotransferase cDNA clone was obtained from a human fetal brain library by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmembrane protein composed of 411 amino acid residues. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, keratan sulfate sulfotransferase activity was overexpressed, but C6ST activity was not increased over that of the control. Structural analysis of 35S-labeled glycosaminoglycan, which was formed from keratan sulfate by the reaction with 35S-labeled 3'-phosphoadenosine 5'-phosphosulfate and the recombinant sulfotransferase, showed that keratan sulfate was sulfated at position 6 of Gal residues. On the basis of the acceptor substrate specificity, we propose keratan sulfate Gal-6-sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotransferase. KSGal6ST was assigned to chromosome 11p11. 1-11.2 by fluorescence in situ hybridization. Among various human adult tissues, a 2.8-kilobase message of KSGal6ST was expressed mainly in the brain. When poly(A)+ RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with about 2.8 kilobases was detected. These observations suggest that KSGal6ST may participate in the biosynthesis of keratan sulfate in the brain and cornea.
Fukuta
M
M
Department of Life Science, Aichi University of Education, Kariya, Aichi 448, Japan.
Inazawa
J
J
Torii
T
T
Tsuzuki
K
K
Shimada
E
E
Habuchi
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000097811:Carbohydrate Sulfotransferases
D018076:DNA, Complementary; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases