Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates.
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.
Bhattacharyya
Sumit
S
Department of Medicine, University of Illinois, Chicago, IL 60612, USA.
Kotlo
Kumar
K
Shukla
Sagar
S
Danziger
Robert S
RS
Tobacman
Joanne K
JK
D000906:Antibodies / Q000737:chemistry; D045744:Cell Line, Tumor; D019765:Chondroitin ABC Lyase / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002811:Chondroitinsulfatases / Q000235:genetics / Q000378:metabolism*; D058575:Decorin; D003871:Dermatan Sulfate / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D020868:Gene Silencing; D006493:Heparin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006497:Heparan Sulfate / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009087:Mucopolysaccharidosis VI / Q000201:enzymology / Q000235:genetics; D020051:N-Acetylgalactosamine-4-Sulfatase / Q000235:genetics / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering; D053668:Syndecan-1 / Q000096:biosynthesis / Q000235:genetics
D000906:Antibodies; D019765:Chondroitin ABC Lyase; D002809:Chondroitin Sulfates; D002811:Chondroitinsulfatases; D058575:Decorin; D003871:Dermatan Sulfate; D016326:Extracellular Matrix Proteins; D006493:Heparin; D006497:Heparan Sulfate; D020051:N-Acetylgalactosamine-4-Sulfatase; D011509:Proteoglycans; D034741:RNA, Small Interfering; D053668:Syndecan-1; C546878:DCN protein, human; C508241:SDC1 protein, human; C102295:GALNS protein, human