Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin. II. - Specific labelling of individual monosaccharides from (U14C)-glucose. Metabolic origin of L-iduronic acid.

1)Individual monosaccharides (uronic acids and aminosugars) have been purified following specific hydrolysis of the mucopolysaccharides from new born rat skin (hyaluronic acid, heparin + heparan sulfate, chondroitin sulfate A, B and C), after incubation with [U14C]-glucose under various conditions and for varying incubation periods. The yields and the specificity of the methods used for hydrolysis are discussed. 2) Monosaccharides from hyaluronic acid and the sulfated mucopolysaccharide fraction are labelled at an approximately equal rate. In addition, high rates of labelling of glucosamine isolated from the sulfated fractions confirms the preferential labelling of (heparin + heparan sulfate) demonstrated with the sulfated polymers. 3) In all fractions, aminosugars are considerably less labelled than the corresponding uronic acids, which suggests the existence of endogeneous diluting precursor pools for the former monosaccharides. 4) No drift of radioactivity from D-glucuronate to L-iduronate could be demonstrated in sulfated mucopolysaccharides after inhibition of their biosynthesis by puromycin or diluting the labelled precursor pools. Hence it has not been possible to substantiate on the surviving tissue the C5 epimerization at the polymer level, as previously demonstrated by other authors with subcellular fractions of various origin.

Synthesis and secretion of sulphated glycosaminoglycans by bovine peridontal ligament fibroblast cultures.

The cultures were allowed to incorporate 35SO2-4 for various periods of time. 35S-labelled macromolecules were isolated from the medium, a trypsin digest of the cells and the cell residue. Ion-exchange chromatography separated the radioactive polysaccharides into heparan sulphate and a galactosaminoglycan population. Most heparan sulphate was in the trypsin digest and cell residue fractions. The galactosaminoglycan fractions were investigated by differential degradations with chondroitinase ABC and AC and ethanol fractionation. The medium galactosaminoglycans contained both glucuronic and iduronic acid residues and existed in copolymeric structures as chondroitin sulphate/dermatan sulphate hybrid molecules. Dermatan sulphate was also detected. In contrast, the trypsin-digest fraction contained mainly chondroitin sulphate-like molecules.

Comparative biochemistry of nucleotide-linked sugars.

Nucleotide-linked sugars have 2 general biochemical functions: they are i) intermediates in the formation of monosaccharides found in complex carbohydrates and ii) glycosyl donors of these monosaccharides. Few sugars arise by reactions not involving nucleotide-linked intermediates. Of these few, glucose, mannose, and N-acetylglucosamine are important in that they are transformed after attachment to nucleotides into most other monosaccharides. Several different nucleotides are involved in these transformations. What factor governs the choice of a particular nucleotide carrier for a given reaction is not apparent, but the use of different nucleotides separates pathways of synthesis and offers a means for their independent control by creating reactions unique to the synthesis of certain products and therefore suitable for regulation. Carrying sugars on different nucleotides may also be advantageous by increasing the accuracy of synthesis of complex carbohydrates. For example, a transferase responsible for the transfer of fucose from GDP-fucose is less likely to transfer galactose from UDP-galactose by mistake than galactose from GDP-galactose. The role of nucleotides in the synthesis of complex carbohydrates thus appears related to the specificity of enzymes that catalyze the modification and transfer of nucleotide-linked sugars.

Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues.

L-[14C]Iduronic acid-containing sulfated galactosaminoglycans were formed by incubation of a fibroblast particulate fraction with UDP-D[14C]glucuronic acid, UDP-N-acetylgalactosamine, and sulfate donor (3'-phosphoadenylylsulfate). The formation of L-iduronic acid was strongly promoted by concomitant sulfation of the polymer. In the absence of sulfate donor 5 to 10% of the [14C]uronic acid residues were L-iduronic acid. However, when 3'-phosphoadenylylsulfate was included in the incubation mixture the amount of L-iduronic acid in the product increased 3 to 5-fold. Furthermore, approximately the same quantity of L-[14C]iduronic acid was recovered from the product formed in a pulse-chase experiment where incorporation of 14C-isotope preceded sulfation. It was therefore concluded that C-5 inversion of D-glucuronic acid to L-iduronic acid occurred on the polymer level as shown previously for the biosynthesis of heparin (Hook, M., Lindahl, U., Backstrom, G., Malmstrom, A., AND Fransson, L-A., J. Biol. Chem. (1974) 249, 3908). This conclusion was supported by the finding that no L[14C]iduronic acid could be detected in the UDP-hexuronic acid pool during this experiment. Nonsulfated and sulfated [14C]galactosaminoglycan products were degraded separately with chondroitinase-AC. The non-sulfated products afforded primarily disaccharide and a small amount of tetrasaccharide, while the sulfated products yielded, in addition, a considerable amount of larger oligosaccharides. Tetrasaccharides from nonsulfated products contained L-iduronic acid indicating that C-5 inversion at solitary sites can occur in the absence of sulfation of adjacent hexosamine moieties. The larger oligosaccharides obtained after chondroitinase-AC digestion of sulfated products yielded L-iduronic acid upon acid hydrolysis and were susceptible to chondroitinase-ABC digestion. The split products were almost exclusively 4-sulfated disaccharides. These results demonstrate that formation of blocks of L-iduronic acid-containing repeat periods is associated with 4-sulfation of adjacent hexosamine moieties.