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

Abstract

1. Two N-acetylgalactosaminyltransferases, designated I and 11, 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-[l -3H]GalNAc in P-glycosidic configuration to the nonreducing terminus of the acceptor substrates GlcA(P 1 -3)Gal(P 1 -3)Gal, GlcA(P 1 -3)Gal(P 1 -4)Glc and GlcA(P 1 -3)Gal.3. Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase 11, which transfers N-acetylgalactosamine from UDP-[I -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-acetylgalactosaminyltransferases I and I1 are substrates of P-Nacetylhexosaminidase, which splits off the transferred [ 1 -3H]GalNAc completely.5. In the microsomal fraction N-acetylgalactosaminyltransferase I1 had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme I1 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 I1 is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.Proteoglycans are polyanionic macromolecules that contain a core protein to which at least one glycosaminoglycan (polysaccharide) chain is covalently linked. These macromolecules are found in almost all mammalin tissues and comprise a wide range of structures involving different core proteins, different classes of glycosaminoglycans and different numbers and lengths of glycosaminoglycan chains (for review see [I]).Proteochondroitin sulfate has been isolated from arterial tissue [2-51 and cultured arterial smooth muscle cells [6] (and references cited therein). The polysaccharide chains of proteochondroitin sulfate are covalently bound to the protein core by a carbohydrate-protein linkage region [l] as shown in the scheme below. The transfer reactions, by which the appropriate glycosyl bonds arise, are designated by the numbers 1 -8. The synthesis of the tetrasaccharide linking the chondroitin sulfate chain to the protein (reactions 1-4) occurs by the catalytic action of specific glycosyltransferases [7 -121, which use the corresponding uridine diphosphate sugars as donor substrates.The glucuronyl transfer to the galactose residue (reaction 4) and the multiple glucuronyl transfer to th...

N-Acetylgalactosamine (GalNAc) Transfer to the Common Carbohydrate-Protein Linkage Region of Sulfated Glycosaminoglycans

“…Although the relationship between the novel ␣-GalNAc T and chondroitin and/or dermatan sulfate biosynthesis is unclear, the observed modulation of the enzyme activity by the sulfated substrates implies that the enzyme might play an important role in the regulation of glycosaminoglycan biosynthesis. Rohrmann et al (21) reported that the ␤-GalNAc transferase (GalNAc T-I), which catalyzes the addition of a GalNAc to the linkage tetrasaccharide, is different from that (GalNAc T-II) which catalyzes the polymerization of a chondroitin sulfate chain. This conclusion was mainly based on the differential thermostability of the two activities, i.e.…”

“…Differential Thermostability of GalNAc Transferases in Serum-Previously, Rohrmann et al (21) proposed that at least two GalNAc transferase activities exist in the microsomal fraction of calf arterial tissue. One, designated GalNAc transferase I (GalNAc T-I), catalyzed transfer of the first GalNAc residue immediately adjacent to the linkage region.…”

Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N -acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

“…Chain polymerization for HS͞Hep is initiated once an ␣-GlcNAc is transferred to this linkage-region tetrasaccharide core by the action of ␣-GlcNAc transferase I (GlcNAcT-I) (9), whereas ␤-GalNAc transfer triggers CS͞DS synthesis (10,11). The first hexosamine transfer, therefore, is considered to be the critical determining step in which the HS͞Hep and CS͞DS chains are selected.…”

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…Then chain elongation occurs by the alternate addition of GalNAc and GlcUA residues. Enzyme activities that catalyze the initiation and elongation processes are termed glycosyltransferase-I and II activities, respectively (12). To date, six glycosyltransferases involved in CS synthesis have been identified (Fig.…”