Glucuronyl C5-epimerases in the biosynthesis of glycosaminoglycans

Sammanfattning: This thesis is focused on two enzymes, both glucuronyl CS-epimerases, that catalyse the conversion of D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) residues in the repeating disaccharide units of heparin/heparan sulphate (HS) and dermatan sulphate (DS) polymers. These glycosaminoglycans are produced as proteoglycans, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions.The reaction catalysed by the C5-epimerases occurs by reversible abstraction and readdition ofa proton at C5 of target hexuronic acid residues, through a carbanion intermediate, with or without inversion of configuration at C5. Studies on the course of C5-3H incorporation from 3H2O into the two hexuronic acid isomers led to proposals for reaction mechanisms. Different incorporation patterns for heparin/HS-epimerase and DS-epimerase implied different sets of bases in the active sites of the two types of enzymes.No significant difference was found between the kinetic behaviour of the heparin-producingmastocytoma enzyme and the heparan sulphate-producing bovine liver enzyme towards differently N-sulphated substrates, suggesting that the enzyme species are functionally similar. The Km values were unexpectedly seen to vary with the enzyme concentration. This effect of enzyme concentration on the Km was also seen for highly purified epimerase and it is likely that the phenomenon is due to the polymeric nature of the substrate.The C5-enimerase involved in the biosynthesis of heparin and HS was cloned from a bovinelung cDNA library and functionally expressed in insect cells, using the baculovirus vector. The cDNA of the cloned enzyme may lack some sequence in the 5'-end, since the cDNA sequence starts in-frame, upstream of the assigned start codon. Northern analysis implicated a ~ 9-kb and a& ~ 5-kb epimerase transcript in both lung and liver tissues, whereas only a ~ 5-kb transcript was seen in mouse mastocytoma cells.