Roberton A M, McKenzie C G, Sharfe N, Stubbs L B
Department of Biochemistry, University of Auckland, New Zealand.
Biochem J. 1993 Aug 1;293 ( Pt 3)(Pt 3):683-9. doi: 10.1042/bj2930683.
A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection.
从结肠中一种降解黏液糖肽的普雷沃氏菌中纯化出了一种新型糖硫酸酯酶。纯化后的酶能从35S标记的天然大鼠胃黏液糖蛋白中去除无机[35S]硫酸盐。黏液糖蛋白的脱硫作用起初很快（10分钟后完成19%），但随后趋于平稳，3小时后仅达到33%。粗制周质提取物可将79%的放射性以无机硫酸盐形式去除。这些结果表明，在没有糖苷酶的情况下，空间位阻可能会限制纯化后的糖硫酸酯酶与黏液糖蛋白寡糖链的接触，和/或该酶对链中一些剩余硫酸化糖的特异性可能有误。根据凝胶过滤色谱法判断，该酶的表观分子量为111 kDa，且似乎由两个相同的亚基组成。该酶定位于细菌的周质中，以猪胃黏液糖肽作为生长底物可显著提高酶水平。酶活性在生长阶段结束时增加。针对低分子量硫酸化分子测试了该酶的底物特异性。6-硫酸葡萄糖和6-硫酸-N-乙酰葡糖胺这两种单糖能迅速脱硫，后者是一些黏液糖蛋白中的主要硫酸化糖。6-硫酸乳糖、6-硫酸半乳糖、硫酸化类固醇以及硫酸软骨素的不饱和二糖硫酸分解产物均未被脱硫。能够从黏液糖蛋白中去除硫酸盐的糖硫酸酯酶可能在消化道中高度硫酸化的黏液糖蛋白降解过程中发挥重要作用，并可能改变黏液糖蛋白在黏膜保护中的功效。
