In vitro polymerization of heparan sulfate backbone by the EXT proteins.
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 3H-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.
Busse
Marta
M
Department of Medical Biochemistry and Microbiology, University of Uppsala, S-751 23 Uppsala, Sweden.
Kusche-Gullberg
Marion
M
D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D020723:Glucuronic Acid / Q000737:chemistry; D016695:Glycosyltransferases / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D000099065:Exostosin 2; D000099058:Exostosin 1
D020723:Glucuronic Acid; D016695:Glycosyltransferases; D006497:Heparan Sulfate; D017351:N-Acetylglucosaminyltransferases; D011134:Polysaccharides; D011994:Recombinant Proteins; D000099065:Exostosin 2; D000099058:Exostosin 1