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

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 Mn 2+ for maximal activity and transfers N ‐acetylgalactosamine residues from UDP‐[1‐ 3 H]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‐ 3 H]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‐ 3 H]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.