Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1
Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols we...
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Published in | The Journal of biological chemistry Vol. 267; no. 10; pp. 6548 - 6555 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
05.04.1992
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Subjects | |
Online Access | Get full text |
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Summary: | Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin
sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various
linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase
followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated
uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose
residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate
esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1
chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from
aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their
linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide
region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first
disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose
in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted,
unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is
demonstrated that separates these reduced disaccharides with exceptional resolution. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(19)50462-8 |