Busse Marta, Kusche-Gullberg Marion
Department of Medical Biochemistry and Microbiology, University of Uppsala, S-751 23 Uppsala, Sweden.
J Biol Chem. 2003 Oct 17;278(42):41333-7. doi: 10.1074/jbc.M308314200. Epub 2003 Aug 7.
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.
多发性外生骨疣是一种常染色体显性遗传性骨疾病，由EXT1和EXT2基因缺陷引起，这两个基因编码参与硫酸乙酰肝素链延长的糖基转移酶。硫酸乙酰肝素的聚合作用是通过将葡萄糖醛酸和N-乙酰葡糖胺单位交替添加到多糖的非还原末端来实现的。EXT1和EXT2被认为是双葡萄糖醛酸基/N-乙酰葡糖胺基转移酶，并且EXT1和EXT2的异源寡聚复合物（EXT1/2）被认为是生物学功能聚合单位。在此，我们研究了重组可溶性EXT1、EXT2和EXT1/2复合物对源自大肠杆菌K5荚膜多糖的外源性寡糖受体的体外聚合能力。重组EXT1或EXT1/2复合物与3H标记的寡糖受体和适当的核苷酸糖一起孵育，导致受体转化为更高分子量的化合物，但EXT1和EXT1/2的效率不同。相比之下，与重组EXT2一起孵育导致添加单个葡萄糖醛酸，但没有进一步的聚合。这些结果表明，单独的EXT1和EXT1/2异源复合物在体外可以作为硫酸乙酰肝素聚合酶，而无需添加额外的辅助蛋白。
