Abstract
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.
Highlights
We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from chick embryo chondrocytes
Materials—The following commercial materials were used: H235SO4 was from DuPont NEN; [3H]NaBH4 (16.3 GBq/mmol) [␣-32P]dCTP (110 TBq/mmol) and Hybond Nϩ were from Amersham Japan, Tokyo; the fetal human brain cDNA library and human multiple tissue Northern blots were from CLONTECH, Palo Alto, CA; unlabeled PAPS, N-acetylglucosamine 6-sulfate, and galactose 6-sulfate were from Sigma; Hiload Superdex 30 HR 16/60, DEAE-Sephadex, and fast desalting column HR 10/10 were from Pharmacia Biotech, Tokyo; chondroitinase ACII, keratanase II, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, and completely desulfated N-resulfated heparin (CDSNS-heparin) were from Seikagaku Corporation, To
Since an amino acid sequence of KSGal6ST deduced from the nucleotide sequence of the cDNA showed 37% homology with chick C6ST, FIG. 7
Summary
Materials—The following commercial materials were used: H235SO4 was from DuPont NEN; [3H]NaBH4 (16.3 GBq/mmol) [␣-32P]dCTP (110 TBq/mmol) and Hybond Nϩ were from Amersham Japan, Tokyo; the fetal human brain cDNA library and human multiple tissue Northern blots were from CLONTECH, Palo Alto, CA; unlabeled PAPS, N-acetylglucosamine 6-sulfate, and galactose 6-sulfate were from Sigma; Hiload Superdex 30 HR 16/60, DEAE-Sephadex, and fast desalting column HR 10/10 were from Pharmacia Biotech, Tokyo; chondroitinase ACII, keratanase II, chondroitin sulfate A (whale cartilage), chondroitin sulfate C (shark cartilage), dermatan sulfate, and completely desulfated N-resulfated heparin (CDSNS-heparin) were from Seikagaku Corporation, To-. Partial Purification of Keratan Sulfate Gal-6-Sulfotransferase—The homogenate of COS-7 cells transfected with pCXNKSGal6ST (224 mg as protein obtained from 80 10-cm dishes) was applied to a DEAESephadex A-50 column (2.2 ϫ 13 cm) equilibrated with buffer A (10 mM Tris-HCl, pH 7.2, containing 20% glycerol, 20 mM MgCl2, 2 mM CaCl2, and 10 mM 2-mercaptoethanol) containing 50 mM NaCl. After the column was washed with 500 ml of the same buffer, the absorbed materials were eluted with 0.5 M NaCl in buffer A. The homogenate of COS-7 cells without transfection obtained from 20 10-cm dishes was separated with DEAE-Sephadex in the same procedures as described above except that the column size, elution volume, and fraction size were reduced to one-fourth. Rate was 1 ml/min, and the column temperature was 40 °C. 0.5-ml fractions were collected
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