Distinct Effects of N-Acetylgalactosamine-4-sulfatase and Galactose-6-sulfatase Expression on Chondroitin Sulfates

Abstract

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) g/mg of protein from baseline of 12.47 (؎0.68) g/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. N-Acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB)2 and galactose-6-sulfatase (GALNS) are two important lysosomal enzymes that hydrolyze sulfate groups from the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and dermatan sulfate. Deficiencies of ASB and GALNS activity are associated with the congenital mucopolysaccharidoses (MPS) MaroteauxLamy syndrome (MPS VI) and Morquio syndrome (MPS IVA), respectively. In these lysosomal storage diseases, there is accumulation of highly sulfated, unmetabolizable glycosaminoglycans and proteoglycans. The impact of sulfatase activity on metabolism of GAGs and the proteoglycans (PGs) with which they are associated has not been widely explored, beyond the congenital storage diseases.Recently, we reported that ASB activity is reduced in uncorrected cystic fibrosis cells, consistent with the accumulation of highly sulfated GAG in pulmonary secretions in cystic fibrosis patients. Following correction of cystic fibrosis transmembrane conductance regulator in pulmonary epithelial cell lines, ASB activity increased significantly (1). Also, we found marked reductions in arylsulfatases A and B, galactose-6-sulfatase, and steroid sulfatase in malignant mammary cell lines (MCF-7, T47D, and HCC 1937), compared with normal primary mammary epi...

Arylsulfatase B regulates colonic epithelial cell migration by effects on MMP9 expression and RhoA activation

“…In mammary cells, we found reduced ASB activity, as well as reduced activity of galactose-6-sulfatase (GALNS), steroid sulfatase (STS), and arylsulfatase A (ARSA), in malignant mammary cells compared to normal cells [2]. In MCF-7 cells, modification of expression of ASB was associated with changes in expression of syndecan and decorin, proteoglycans with N-acetylgalactosamine-4-sulfate residues as part of their sulfated glycosaminoglycan (sGAG) component [3]. Reduction of ASB activity was also associated with deficiency of CFTR in cystic fibrosis (CF) cell lines [4], and reduced ASB activity was found in peripheral blood smears from 57 children with CF, compared to 181 control patients [5].…”

“…Cross-reactivity of the anti-C4S with CS-E or C6S was excluded by similar tests. Immunoprecipitation was performed as described previously [3].…”

Increased Expression of Colonic Wnt9A through Sp1-mediated Transcriptional Effects involving Arylsulfatase B, Chondroitin 4-Sulfate, and Galectin-3

“…Total sulfated glycosaminoglycans, including C4S, chondroitin 6-sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, and heparin, were measured in NCM460 cells, HT-29 cells, mouse colon following exposure to carrageenan, and colon of ARSB-null mice by sulfated glycosaminoglycan (GAG) assay (Blyscan TM , Biocolor, Ltd., Newtownabbey, Northern Ireland) as described previously (32). C4S was determined following immunoprecipitation of cell or tissue lysates with C4S antibody (4D1, Abnova, Littleton, CO or LY111 Amsbio, LLC, Lake Forest, CA).…”

Extra-Lysosomal Localization of Arylsulfatase B in Human Colonic Epithelium

“…ARSB activity was measured in the frozen tissue samples following an established method that used the substrate 4-methyumbilliferyl sulfate (Bhattacharyya and Tobacman 2007;Bhattacharyya et al 2008;Bhattacharyya, Solakyildirim, et al 2010). Then, 20 µl of tissue homogenate and 80 µl of assay buffer (0.05M Na acetate buffer, pH 5.6) were combined with 100 µl of substrate (5 mM 4-MUS in assay buffer) in wells of a microplate.…”

“…Recent published work demonstrated that reduced ARSB activity is associated with cystic fibrosis, in which there is accumulation of sulfated polysaccharides, including the sulfated glycosaminoglycans chondroitin sulfate and dermatan sulfate (Bhattacharyya and Tobacman 2007;Bhattacharyya et al 2008;Bhattacharyya, Solakyildirim, et al 2010). Immunohistochemistry and confocal microscopy have shown prominent cell membrane immunostaining for ARSB in human colonic and bronchial epithelial cells and cerebrovascular cells, as well as enzymatic activity in the cell membrane fraction (Bhattacharyya, Solakyildirim, et al 2010;Tobacman 2006, 2009).…”