Lacoste C H, Freeze H H, Jones J A, Kaplan A
Department of Microbiology, St Louis University School of Medicine, Missouri 63104.
Arch Biochem Biophys. 1989 Sep;273(2):505-15. doi: 10.1016/0003-9861(89)90510-9.
Lysosomal enzymes from Dictyostelium discoideum contain unusual sulfated N-linked oligosaccharides, whose synthesis has been well studied in vivo. However, little is known about the properties of the pertinent sulfotransferases. To study these transferases, we have prepared a cell-free system which transfers 35SO4 from 3'-phosphoadenosine 5'-phosphosulfate to either endogenous or exogenous acceptors. We found that the 35SO4 was released from macromolecules by protein N-glycanase F to yield a mixture of anionic oligosaccharides with 1-6 negative charges. Some of the labeled molecules contained acid-stable methyl phosphodiesters but none contained phosphomoesters or acid-labile diesters. The sulfate was found in molecules with the acid stability characteristic of esters of primary alcohols. In all these ways, the products resembled those generated in vivo. We also demonstrated that a membrane-associated form of beta-hexosaminidase and the precursor of alpha-mannosidase were among the products. In addition, glycoproteins prepared from a sulfation-deficient mutant strain could act as exogenous acceptors in permeabilized vesicles.
盘基网柄菌的溶酶体酶含有不寻常的硫酸化N-连接寡糖，其合成在体内已得到充分研究。然而，对于相关硫酸转移酶的特性却知之甚少。为了研究这些转移酶，我们制备了一种无细胞系统，该系统可将3'-磷酸腺苷5'-磷酸硫酸酯中的35SO4转移至内源性或外源性受体。我们发现，35SO4可通过蛋白N-聚糖酶F从大分子中释放出来，产生带有1至6个负电荷的阴离子寡糖混合物。一些标记分子含有酸稳定的甲基磷酸二酯，但均不含磷酸单酯或酸不稳定的二酯。硫酸盐存在于具有伯醇酯酸稳定性特征的分子中。所有这些方面，产物都类似于体内产生的产物。我们还证明，β-己糖胺酶的膜结合形式和α-甘露糖苷酶的前体也在产物之中。此外，从硫酸盐缺乏突变株制备的糖蛋白可作为通透化囊泡中的外源性受体。
