Structure and Anticoagulant Activity of a Fucosylated Chondroitin Sulfate from Echinoderm

doi:10.1074/jbc.271.39.23973

Structure and Anticoagulant Activity of a Fucosylated Chondroitin Sulfate from Echinoderm

SULFATED FUCOSE BRANCHES ON THE POLYSACCHARIDE ACCOUNT FOR ITS HIGH ANTICOAGULANT ACTION*

From the Departamento de Bioquímica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil
From the National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, EN6 3QG United Kingdom
From the Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biophysics, Washington University, School of Medicine, St. Louis, Missouri 63110
From the Department of Medicine, Aker University Hospital, Oslo, N-0514 Norway

^‡To whom correspondence should be addressed. Fax: 55-21-270-8647; E-mail: MOURÃO{at}SERVER.BIOQMED.UFRJ.BR

Abstract

A polysaccharide isolated from the body wall of the sea cucumber Ludwigothurea grisea has a backbone like that of mammalian chondroitin sulfate: [4-β-D-GlcA-1→3-β-D-GalNAc-1]_n but substituted at the 3-position of the β-D-glucuronic acid residues with sulfated α-L-fucopyranosyl branches (Vieira, R. P., Mulloy, B., and Mourão, P. A. S. (1991) J. Biol. Chem. 266, 13530-13536). Mild acid hydrolysis removes the sulfated α-L-fucose branches, and cleaved residues have been characterized by ¹H NMR spectroscopy; the most abundant species is fucose 4-O-monosulfate, but 2,4- and 3,4-di-O-sulfated residues are also present. Degradation of the remaining polysaccharide with chondroitin ABC lyase shows that the sulfated α-L-fucose residues released by mild acid hydrolysis are concentrated toward the non-reducing end of the polysaccharide chains; enzyme-resistant polysaccharide material includes the reducing terminal and carries acid-resistant L-fucose substitution. The sulfated α-L-fucose branches confer anticoagulant activity on the polysaccharide. The specific activity of fucosylated chondroitin sulfate in the activated partial thromboplastin time assay is greater than that of a linear homopolymeric α-L-fucan with about the same level of sulfation; this activity is lost on defucosylation or desulfation but not on carboxyl-reduction of the polymer. Assays with purified reagents show that the fucosylated chondroitin sulfate can potentiate the thrombin inhibition activity of both antithrombin and heparin cofactor II.

Footnotes

↵* This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT and PADCT), Financiadora de Estudos e Projetos (FINEP), Mizutani Foundation for Glycoscience (to P. A. S. M.), and the International Foundation for Science (to M. S. G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

APTT

activated partial thromboplastin time.
↵²Incubation with chondroitin AC lyase produces similar results as obtained with chondroitin ABC lyase.
↵³Comparison of electrophoretic behavior of the native branched compound with the defucosylated, relatively unbranched molecule (Fig. 3B) does not indicate molecular weight change with sufficient accuracy to allow us to exclude other modifications, such as a small extent of cleavage of N-acetyl-β-D-galactosamine glycosidic bonds in the polysaccharide, during defucosylation by mild acid hydrolysis. However, our proposed structure of the fucosylated chondroitin sulfate remains valid except in the highly unlikely circumstance that the mild acid hydrolysis cleaves a single galactosamine glycosidic linkage in each molecule, separating blocks of the polysaccharide containing chondroitin lyase-resistant and sensitive portions.
↵⁴Apparently L-fucose residues removed during the desulfation reaction are evenly distributed along the polysaccharide since no disaccharide is produced by chondroitin lyase digestion of the desulfated polymer (7), in contrast with results from digestion of the partially defucosylated material produced by mild acid hydrolysis (Fig. 3).
↵⁵Preliminary experiments suggest that the fucosylated chondroitin sulfate has no effect on the bleeding time when the scarified rat tail was placed in a polysaccharide solution up to concentration of 1.0 mg/ml.
- Received March 14, 1996.
- Revision received June 4, 1996.