A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase.
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.
Ohtake
Shiori
S
Department of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan.
Kimata
Koji
K
Habuchi
Osami
O
D000818:Animals; D001717:Birds; D015153:Blotting, Western; D019556:COS Cells; D019765:Chondroitin ABC Lyase / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002810:Chondroitinases and Chondroitin Lyases / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D018076:DNA, Complementary / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D005966:Glucuronidase / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014312:Trisaccharides / Q000737:chemistry; D014574:Uronic Acids / Q000737:chemistry
D018076:DNA, Complementary; D004187:Disaccharides; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; D014574:Uronic Acids; D010724:Phosphoadenosine Phosphosulfate; D002809:Chondroitin Sulfates; C416670:N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; D015238:Sulfotransferases; D005966:Glucuronidase; D002810:Chondroitinases and Chondroitin Lyases; D019765:Chondroitin ABC Lyase; D008628:Mercury; C029823:mercuric acetate