Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate

We identified a novel human chondroitin N-acetyl-galactosaminyltransferase, designated chondroitin Gal-NAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GaINAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type 11 transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GaINAcT-1. Like chondroitin GaINAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred 131,4-N-acetylgalactosamine (GaINAc) from UDP-[H-3]GaINAc to a polymer chondroitin representing growing chondroitin chains (beta-GaINAc transferase II activity) but also to GleUAbeta1-3Galbeta1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-Gal-NAc transferase I that transfers the first GaINAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GIcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GaINAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GaIN-AcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GaINAcT-1. Thus, we demonstrated redundancy in the chondroitin GaINAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglyeans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.