Abstract
N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO(4)) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO(4)) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.
Highlights
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EBI Data Bank with accession numbers AB062423 and AB062424
Materials—The following commercial materials were used: H235SO4 was from PerkinElmer Life Sciences; chondroitinase ACII, chondroitinase ABC, chondro-6-sulfatase, CS-A, CS-C, DS, heparan sulfate, completely desulfated N-resulfated heparin (CDSNS-heparin), ⌬Di-0S, ⌬Di-6S, ⌬Di-4S, ⌬Di-diSD, and ⌬Di-diSE were from Seikagaku Corporation, Tokyo; Partisil-10 SAX was from Whatman; unlabeled PAPS, N-acetylgalactosamine 4-sulfate, N-acetylgalactosamine 6-sulfate, N-acetylgalactosamine 4,6-bissulfate, hyaluronidase, -glucuronidase and 2-acetamido-2-deoxy-D-galactonic acid-1,4-lactone were from Sigma; Hiload Superdex 30 HR 16/60 and Fast Desalting Column HR 10/10 were from Amersham Pharmacia Biotech; fresh squids, Ommastrephes sloani pacificus, were obtained locally
When primer 5s-1 and 4a-2 designed from the amino acid sequences of peptides 5 and 4, respectively, were used in a PCR with poly(A)ϩ RNA from squid cartilage as a template, no amplification of DNA was observed; a DNA fragment of about 380 base pairs was clearly amplified on the second PCR, in which the reaction mixture of the first PCR was used as a template and oligonucleotide 5s-2 and 4a-1 were used as primers (Fig. 1B, lane 1)
Summary
Materials—The following commercial materials were used: H235SO4 was from PerkinElmer Life Sciences; chondroitinase ACII, chondroitinase ABC, chondro-6-sulfatase, CS-A (whale cartilage), CS-C (shark cartilage), DS (pig skin), heparan sulfate (bovine kidney), completely desulfated N-resulfated heparin (CDSNS-heparin), ⌬Di-0S, ⌬Di-6S, ⌬Di-4S, ⌬Di-diSD, and ⌬Di-diSE were from Seikagaku Corporation, Tokyo; Partisil-10 SAX was from Whatman; unlabeled PAPS, N-acetylgalactosamine 4-sulfate, N-acetylgalactosamine 6-sulfate, N-acetylgalactosamine 4,6-bissulfate, hyaluronidase (bovine testis), -glucuronidase (bovine liver, type B-3) and 2-acetamido-2-deoxy-D-galactonic acid-1,4-lactone were from Sigma; Hiload Superdex 30 HR 16/60 and Fast Desalting Column HR 10/10 were from Amersham Pharmacia Biotech; fresh squids, Ommastrephes sloani pacificus, were obtained locally. Determination of Amino Acid Sequence of the Purified Protein—The squid GalNAc4S-6ST was purified from the squid cartilage as described previously [29]. The first strand of cDNA was synthesized by the reverse transcriptase reaction using poly(A)ϩ RNA from the squid cartilage as a template and oligo(dT) or random oligonucleotide as a primer. The PCR reaction was carried out in a final volume of 25 l containing 25 pmol each of the oligonucleotide primers (5s-1 and 4a-2), 1 l of the reverse transcriptase reaction mixture in which the first strand cDNA was synthesized, 0.2 mM each of four deoxynucleoside triphosphates, and 1.5 units of Taq polymerase (Qiagen). After the reaction was stopped, 35S-labeled glycosaminoglycans were isolated by the precipitation with ethanol followed by gel chromatography with a fast desalting column as described previously [39] and radioactivity was determined.
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