title: Chloroquine reduces arylsulphatase B activity and increases chondroitin-4-sulphate: implications for mechanisms of action and resistance.
abstract: BACKGROUND
  The receptors for adhesion of Plasmodium falciparum-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB.
  
  
  METHODS
  Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the in vitro exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using in vitro assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure.
  
  
  RESULTS
  In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization.
  
  
  CONCLUSIONS
  The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of P. falciparum-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.
authors.affiliation: Department of Medicine, University of Illinois at Chicago, 840 S, Wood St,, CSN 440 M/C 718 Chicago, IL 60612, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Kemal
authors.initials: K
authors.lastname: Solakyildirim
authors.forename: Zhenqing
authors.initials: Z
authors.lastname: Zhang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.forename: Joanne K
authors.initials: JK
authors.lastname: Tobacman
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D001192:Arylsulfatases / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology*; D002809:Chondroitin Sulfates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003600:Cytosol / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods
chemical_list: D000962:Antimalarials; D004791:Enzyme Inhibitors; D002738:Chloroquine; D002809:Chondroitin Sulfates; D001192:Arylsulfatases