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Xylosyl transfer to the core protein precursor of the rat chondrosarcoma proteoglycan

Lohmander, Stefan LU ; Shinomura, T.; Hascall, V. C. and Kimura, J. H. (1989) In Journal of Biological Chemistry 264(31). p.18775-18780
Abstract

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography,... (More)

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

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published
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in
Journal of Biological Chemistry
volume
264
issue
31
pages
6 pages
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • Scopus:0024414803
ISSN
0021-9258
language
English
LU publication?
yes
id
52f6a2c0-7074-42f4-ae84-0c0dbf6711b3
date added to LUP
2016-05-04 18:14:26
date last changed
2016-07-04 12:12:28
@misc{52f6a2c0-7074-42f4-ae84-0c0dbf6711b3,
  abstract     = {<p>Rat chondrosarcoma chondrocytes were labeled with [<sup>3</sup>H]serine or [<sup>3</sup>H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [<sup>3</sup>H]mannose to [<sup>3</sup>H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.</p>},
  author       = {Lohmander, Stefan and Shinomura, T. and Hascall, V. C. and Kimura, J. H.},
  issn         = {0021-9258},
  language     = {eng},
  number       = {31},
  pages        = {18775--18780},
  publisher    = {ARRAY(0x7bea130)},
  series       = {Journal of Biological Chemistry},
  title        = {Xylosyl transfer to the core protein precursor of the rat chondrosarcoma proteoglycan},
  volume       = {264},
  year         = {1989},
}