Heparan, the common unsulfated precursor of
heparan sulfate (HS) and
heparin, is synthesized on the
glycosaminoglycan-
protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core
proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in
hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit
GlcNAc transferase and GlcUA
transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant
enzymes. Here we report in vitro HS polymerization. Recombinant soluble
enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan
polymers with average molecular weights greater than 1.7 x 105 using
UDP-[3H]GlcNAc and
UDP-GlcUA as donors on the recombinant
glypican-1 core
protein and also on the synthetic linkage region analog GlcUA-Gal-O-C2H4NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time
proteoglycan, alpha-
thrombomodulin, that is normally modified with
chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as
N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core
protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization.