Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate

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Abstract
1 Two N-acetylgalactosaminyltransferases, designated I and II, have been purified from the microsomal fraction of calf arterial tissue and separated on Bio-Gel A. 2 N-Acetylgalactosaminyltransferase I was purified 450-fold. It requires Mn2+ for maximal activity and transfers N-acetylgalactosamine residues from UDP-[1-3H]GalNAc in β-glycosidic configuration to the non-reducing terminus of the acceptor substrates GlcA(β1–3)Gal(β1–3)Gal, GlcA(β1–3)Gal(β1–4)Glc and GlcA(β1–3)Gal. 3 Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase II, which transfers N-acetylgalactosamine from UDP-[1-3H]GalNAc to the non-reducing glucuronic acid residues of oligosaccharide acceptor substrates. Maximum transfer rates were obtained with a decasaccharide derived from chondroitin. Longer or shorter-chain chondroitin oligosaccharides are less effective acceptor substrates. 4 All reaction products formed by N-acetylgalactosaminyltransferase I and II are substrates of β-N-acetylhexosaminidase, which splits off the transferred [1-3H]GalNAc completely. 5 In the microsomal fraction N-acetylgalactosaminyltransferase II had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme II loses much of its activity during the purification procedure and undergoes rapid thermodenaturation. 6 GlcA-Gal-Gal is a characteristic sequence of the carbohydrate-protein linkage region of proteochondrioitin sulfate. The acceptor capacity of this trisaccharide suggests that N-acetylgalactosaminyltransferase I is involved in the synthesis of the carbohydrate-protein linkage region. Since N-acetylgalactosaminyltransferase II is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.