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Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts

Fransson, L Å; Karlsson, P and Schmidtchen, Artur LU (1992) In Biochimica et Biophysica Acta 1137(3). p.287-297
Abstract
(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception:... (More)
(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous beta-D-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Skin, Human, Fibroblast, In vitro, Proteoglycan, Biosynthesis, Culture medium, Metabolic inhibitor, Glycoproteins
in
Biochimica et Biophysica Acta
volume
1137
issue
3
pages
287 - 297
publisher
Elsevier
external identifiers
  • pmid:1445930
  • scopus:0026495312
ISSN
0006-3002
language
English
LU publication?
yes
id
60e8c921-ef64-4f29-8560-b006145a228b (old id 1106754)
date added to LUP
2008-08-01 14:42:12
date last changed
2017-02-26 04:20:07
@article{60e8c921-ef64-4f29-8560-b006145a228b,
  abstract     = {(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous beta-D-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.},
  author       = {Fransson, L Å and Karlsson, P and Schmidtchen, Artur},
  issn         = {0006-3002},
  keyword      = {Skin,Human,Fibroblast,In vitro,Proteoglycan,Biosynthesis,Culture medium,Metabolic inhibitor,Glycoproteins},
  language     = {eng},
  number       = {3},
  pages        = {287--297},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta},
  title        = {Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts},
  volume       = {1137},
  year         = {1992},
}