Effect of brefeldin A on heparan sulphate biosynthesis in Madin-Darby canine kidney cells.
Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin-Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.
Kolset
Svein Olav
SO
Institute for Nutrition Research, University of Oslo, P.O. Box 1046 Blindern, 0316 Oslo, Norway. s.o.kolset@basalmed.uio.no
Prydz
Kristian
K
Fjeldstad
Katja
K
Safaiyan
Fariba
F
Vuong
Tram Thu
TT
Gottfridsson
Eva
E
Salmivirta
Markku
M
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D002460:Cell Line; D004187:Disaccharides / Q000737:chemistry; D004285:Dogs; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007668:Kidney; D013431:Sulfates / Q000378:metabolism; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism
D020126:Brefeldin A; D004187:Disaccharides; D005944:Glucosamine; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D013431:Sulfates