Oversulfated chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid structures bind growth factors, promote the neurite outgrowth of hippocampal neurons in vitro, and have been implicated in the development of the brain. To investigate the expression of functional oversulfated DS structures in the brain, a novel monoclonal antibody (mAb), 2A12, was generated against DS (An-DS) from ascidian Ascidia nigra, which contains a unique iD disaccharide unit, iduronic acid (2-O-sulfate)␣133GalNAc(6-O-sulfate), as a predominant disaccharide. mAb 2A12 specifically reacted with the immunogen, and recognized iD-enriched decasaccharides as minimal structures. The 2A12 epitope was specifically observed in the hippocampus and cerebellum of the mouse brain on postnatal day 7, and the expression in the cerebellum disappeared in the adult brain, suggesting a spatiotemporally regulated expression of this epitope. Embryonic hippocampal neurons were immunopositive for 2A12, and the addition of the antibody to the culture medium significantly reduced the neurite growth of hippocampal neurons. In addition, two minimum 2A12-reactive decasaccharide sequences with multiple consecutive iD units were isolated from the An-DS chains, which exhibited stronger inhibitory activity against the binding of various growth factors and neurotrophic factors to immobilized embryonic pig brain CS/DS chains (E-CS/DS) than the intact E-CS/DS, suggesting that the 2A12 epitope at the neuronal surface acts as a receptor or co-receptor for these molecules. Thus, we have selected a unique antibody that recognizes iD-enriched oversulfated DS structures, which are implicated in the development of the hippocampus and cerebellum in the central nervous system. The antibody will also be applicable for investigating structural alterations in CS/DS in aging and pathological conditions.
Chondroitin sulfate (CS)1 and dermatan sulfate (DS), as well as heparan sulfate (HS), are glycosaminoglycans, which are synthesized as carbohydrate side chains covalently attached to a core protein of proteolgycan (PG) (for reviews, see Ref. 1-3). CS/DS-PGs are present at cell surfaces and in the extracellular matrices of most tissues, and are significant components in the mammalian brain, where they participate in neural development by regulating neuronal adhesion and migration, neurite formation, and axonal guidance (for reviews, see . The backbone of CS and DS consists of repeating disaccharide units of -GlcUA-GalNAc-and -IdoUA-GalNAc-, respectively, and hybrid chains composed of both units in varying proportions also exist (9). These disaccharide units are modified during chain elongation by specific sulfotransferases at C-2 of GlcUA/IdoUA and/or C-4 and/or C-6 of GalNAc in various combinations, thereby producing characteristic sulfation patterns and enormous structural diversity for CS, DS, and CS/DS hybrid chains. Recent studies have shown that the GlcUA/IdoUA ratio and sulfation pattern of the brain CS/DS change during development (10 -12), and subpopulations of the bra...