J. Biochem, 2003, Vol. 133, No. 6 783-790
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Enhancement of Neurite Outgrowth–Promoting Activity by Heparin Derivatives in Sodium Chlorate–Treated Explant Cultures of Rat Central Neurons
1 Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashiyamato, Tokyo 207-0021; 2 Institute of Zoology, The University of Hohenheim, Garbenstr. 30, 70593 Stutgart, Germany; and 3 Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513
Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH. Digestion of OR2DSH with heparitinases generated unsaturated disaccharides, comprising 86.5% 
DiHS-(6,N)S (UA1
4GlcNS(6S)) and 13.5% DiHS-NS (UA14GlcNS), as well as undigested oligosaccharides in which uronate moieties were derivatized by the cleavage of the covalent bond between the C-2 and C-3 positions by periodate-oxidation. The molecular mass of OR2DSH was determined to be 11 kDa, which is almost the same as those of other heparin derivatives such as 2-O-desulfated heparin (2DSH), 6-O-desulfated heparin (6DSH) and N-desulfated N-reacetylated heparin (NDSNAc-heparin). The ability of OR2DSH to enhance neurite outgrowth-promoting activity was evaluated using the explant culture of neocortical tissue from rat embryo in which endogenous heparan sulfate at the cell surface lost substantial numbers of sulfate groups by the action of 40 µM sodium chlorate. The maximum activity of OR2DSH (29.7%) was achieved at 10 µg/ml, and those of OR-heparin (21.7%), 2DSH (18.7%) and intact heparin (16.3%) were 100 µg/ml, whereas that of NDSNAc-heparin (16.5%) was 1,000 µg/ml. Completely 6-O-desulfated heparin (100:6DSH) exhibited very weak activity (3.3%) at 1,000 µg/ml. These results suggest that the potency of OR2DSH to enhance neurite outgrowth–promoting activity is exerted synergetically by two different components in OR2DSH, i.e., the IdoA 
14GlcNS(6S) unit, which contains 6-O- and 2-N-sulfate groups, and the uronate moiety in which the covalent bond between C-2 and C-3 is cleaved, although the mode of action remains to be clarified.
+ Present address: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA30912 Georgia, USA.
To whom correspondence should be addressed. Tel: +81-42-563-5823, Fax: +81-42-563-5848, E-mail: kariya{at}seikagaku.co.jp