Bhattacharyya Sumit, Kotlo Kumar, Shukla Sagar, Danziger Robert S, Tobacman Joanne K
Department of Medicine, University of Illinois, Chicago, IL 60612, USA.
J Biol Chem. 2008 Apr 11;283(15):9523-30. doi: 10.1074/jbc.M707967200. Epub 2008 Feb 18.
The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.
硫酸酯酶，即N - 乙酰半乳糖胺 - 4 - 硫酸酯酶（芳基硫酸酯酶B（ASB））和半乳糖 - 6 - 硫酸酯酶（GALNS）可水解硫酸软骨素（CS）的硫酸基团。ASB和GALNS的缺乏与黏多糖贮积症相关。为了确定ASB和GALNS的表达是否会对糖胺聚糖（GAGs）和蛋白聚糖产生影响，而不仅仅是与黏多糖贮积症相关，我们在MCF - 7细胞中通过过表达以及用小干扰RNA沉默的方式来改变ASB和GALNS的表达。在用C4S和CS抗体进行免疫沉淀并用软骨素酶ABC处理后，测量总硫酸化GAG（sGAG）、硫酸软骨素4 - 硫酸酯（C4S）和总硫酸软骨素（CSs）的含量。在沉默ASB或GALNS后，总sGAG、C4S和CS显著增加。在ASB或GALNS过表达后，总sGAG、C4S和CS显著下降。用软骨素酶ABC处理细胞裂解物后的测量结果表明，包括肝素、硫酸乙酰肝素、硫酸皮肤素和硫酸角质素在内的其他sGAG的含量没有变化。在ASB过表达并用C4S抗体进行免疫沉淀后，几乎检测不到sGAG。在ASB沉默后，总sGAG含量从基线的12.47（±0.68）μg/mg蛋白质增加到23.39（±1.06）μg/mg蛋白质。在ASB和GALNS过表达后，含CS的蛋白聚糖核心蛋白syndecan - 1和核心蛋白聚糖的mRNA表达显著上调。可溶性syndecan - 1蛋白在ASB和GALNS增加后升高，在沉默后降低，与CS的变化相反。这些发现表明，溶酶体硫酸酯酶ASB和GALNS表达的改变可调节CSs的含量。
