In Vitro Polymerization of Heparan Sulfate Backbone by the EXT Proteins

Abstract

Multiple exosotoses is a dominantly inherited bone disorder caused by defects in EXT1 and EXT2, genes encoding glycosyltransferases involved in heparan sulfate chain elongation. Heparan sulfate polymerization occurs by the alternating addition of glucuronic acid and N-acetylglucosamine units to the nonreducing end of the polysaccharide. EXT1 and EXT2 are suggested to be dual glucuronyl/N-acetylglucosaminyltransferases, and a heterooligomeric complex of EXT1 and EXT2 (EXT1/2) is considered to be the biological functional polymerization unit. Here, we have investigated the in vitro polymerization capacities of recombinant soluble EXT1, EXT2, and EXT1/2 complex on exogenous oligosaccharide acceptors derived from Escherichia coli K5 capsular polysaccharide. Incubations of recombinant EXT1 or EXT1/2 complex with 3 H-labeled oligosaccharide acceptors and the appropriate nucleotide sugars resulted in conversion of the acceptors to higher molecular weight compounds but with different efficacies for EXT1 and EXT1/2. In contrast, incubations with recombinant EXT2 resulted in the addition of a single glucuronic acid but no further polymerization. These results indicate that EXT1 alone and the EXT1/2 heterocomplex can act as heparan sulfate polymerases in vitro without the addition of additional auxiliary proteins.

Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

“…EXT2 is necessary for HS polymerization as evident from mutational analysis in Drosophila, mouse, and zebrafish orthologs of EXT2 (30 -34). Previous in vitro data indicate that EXT1 has readily detectable GlcA and GlcNAc transferase activities, whereas the enzyme activities of EXT2 are less manifest (7,8,13). The lack of effect of EXT2 overexpression could be due to the fact that EXT2 may have a different function in HS polymerization from EXT1.…”

“…The expressed proteins were affinity captured on anti-FLAG-agarose, and the bound fusion proteins were analyzed for glycosyltransferase activities. The glycosyltransferase activities of immunopurified EXT1, EXT2, and EXT1/EXT2 have been described previously (13). The immunopurified EXTL3 exhibited weak GlcNAc-TII activity and no detectable GlcA-TII activity (data not shown).…”

“…Glycosyltransferase Assays-GlcNAc and GlcA transferase activities were measured essentially as described (13). GlcNAc-T activities of crude cell extracts were determined by incubating 60 -90 g of lysate protein with 0.125 Ci of UDP-[ 14 C]GlcNAc and 50 g of [GlcA-GlcNAc] n oligosaccharide (the reducing terminal sugar is modified to an 2,5-anhydromannitol unit).…”

“…Even though EXT1 alone is able to polymerize the HS backbone structure in vitro, all evidence suggests that both EXT1 and EXT2 are necessary for chain elongation (13,14). All three EXTLs catalyze GlcNAc transferase reactions.…”

Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

“…The EXT1/EXT2 heterooligomer has a much higher glycosyltransferase activity than EXT1 alone, suggesting that this complex represents the biologically relevant form of the HS polymerization unit (12,13). EXT1 alone, and the EXT1/EXT2 complex, can catalyze in vitro polymerization of the HS backbone structure on an oligosaccharide primer, whereas the activity of EXT2 is much weaker (14). Interactions have also been demonstrated between the C5-epimerase and the 2-O-sulfotransferase (15) and between the xylosyltransferase and galactosyltransferase-I (16,17).…”