Study on the biological function of glycoconjugates

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(1) We cloned mouse and human chondroitin 4-sulfotransferase gene, characterized the protein expressed from the gene and determined expression pattern in various tissues. We also mapped the gene in human chromosome.(2) We cloned and characterized human GalNAc 4-sulfotransferase that strongly expressed in human pituitary grand. We also cloned mouse GalNAc 4-sulfotransferase I and II and determined in various tissues by Northern blot and RT-PCR.(3) We expressed NSIST, which was cloned from Torpedo electric organ, in COS-7 cells and found that substrate specificity of NSIST was very similar to that of chondroitin 6-sulfotransferase.(4) Af-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST)was purified to apparent homogeneity from the squid cartilage. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the i … More nternal Af-acetylgalactosamine 4-sulfate residues. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of W-acetylgalactosamine residues were used as acceptors, efficient sulfation of position 6 at nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed. We cloned and characterized 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 PCR, and 3) homology search using the amino acid sequence deduced from the squid DNA. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from PAPS 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 RAG-associated gene. Less