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) 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.
Highlights
N-Acetylgalactosamine-4-sulfatase2 and galactose-6-sulfatase (GALNS) are two important lysosomal enzymes that hydrolyze sulfate groups from the glycosam
Transfection of MCF-7 cells with 2 g of GALNS increased the GALNS activity in the cells to 8.15 (Ϯ0.20), a 117% increase, and statistically significant compared with controls (p Ͻ 0.001, one-way analysis of variance and Tukey-Kramer post-test)
The intracellular lysosomal enzymes ASB and GALNS have been associated with the occurrence of the mucopolysaccharidoses or lysosomal storage diseases
Summary
Sulfotransferases have been regarded as the predominant intracellular mechanism for the modification of the extent of sulfation of the GAG, the effects of extracellular sulfatase enzymes have been recognized. The mammalian sulfatases (Sulf and Sulf2) are secreted and act as specific heparin 6-O-sulfatases [10, 11] They can mediate heparin-binding growth factor signaling by modification of GAG chains extracellularly [12]. In this report we present distinct effects of the activity of the lysosomal sulfatases ASB and GALNS on modification of CSs in MCF-7 cells, hypothesizing a role for these enzymes in cellular metabolism, beyond that associated with the mucopolysaccharidoses. To clarify if modification of lysosomal sulfatase expression can affect chondroitin sulfations in cells unaffected by genetic deficiency of sulfatase activity, we have considered the effects of silencing and overexpression of the lysosomal enzymes ASB and GALNS on total sulfated GAG (sGAG), total CS (C4S, C6S, CS-D, and CS-E), and chondroitin 4-sulfate (C4S) content, in MCF-7 cells in tissue culture. We have determined the effects of modification of sulfatase enzymes on syndecan-1 and decorin mRNA expression and soluble syndecan-1 protein, because ASB and GALNS can hydrolyze the sulfate groups of the chondroitin component of the PG formed by these core proteins
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