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Glycobiology Advance Access published online on April 17, 2003
Glycobiology, doi:10.1093/glycob/cwg074
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Submitted on January 27, 2003
Revised on March 26, 2003
Accepted on March 27, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Nicola Volpi 1*

1 Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy

* To whom correspondence should be addressed. E-mail: volpi{at}unimo.it.

Abstract

Escherichia coli K4 bacterium synthesizes a nonsulphated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid ({beta}1->3) and N-acetyl-D-galactosamine ({beta}1->4) to which {beta}-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid ({beta}1->3) and N-acetyl-D-galactosamine ({beta}1->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg of K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform-size K4 and K4d oligosaccharide species, from approx. 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, ->4)-GlcUA-{beta}(1->3)GalNAc-{beta}(1->, will be available for investigating important biological functions of this polymer.


K4 polysaccharide, Chondroitin, Glycosaminoglycans, Oligosaccharides
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