A Functional Dermatan Sulfate Epitope Containing Iduronate(2-O-sulfate)α1–3GalNAc(6-O-sulfate) Disaccharide in the Mouse Brain

Abstract

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...

Synthetic sugars enhance the functional glycomics toolkit

“…The neurite outgrowth studies confirm the results obtained by Bao et al using libraries of natural saccharides 7 . Both groups also use blocking antibodies against selected epitopes 2,7 , and these tools should prove very useful for investigating epitope expression and function in vivo.…”

“…The neurite outgrowth studies confirm the results obtained by Bao et al using libraries of natural saccharides 7 . Both groups also use blocking antibodies against selected epitopes 2,7 , and these tools should prove very useful for investigating epitope expression and function in vivo. The new study demonstrates that integration of diverse experimental platforms provides an effective approach for investigating the ways in which GAGs encode functional information in a sequence-specific manner 2 .…”

“…The authors focused on a small set of saccharides anticipated to effect neurite outgrowth based on previous studies involving natural structures 7 . The functionalized sugars were synthesized by highly convergent chemistry that allowed for incorporation of specific sulfation sequences on these carbohydrate templates as well as stereocontrol of the glycosylation reactions.…”

Demonstration of the Pleiotrophin-binding Oligosaccharide Sequences Isolated from Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains of Embryonic Pig Brains

“…These octasaccharides are derived from the corresponding parent decasaccharide sequences flanked by two IdoUA residues in E-CS/DS (TABLE THREE) as in the case of the PTN-unbound octasaccharides (TABLE TWO). Notably, we have now verified iD units, whose existence in the mouse cerebellum was previously suggested based on the immunostaining with the iD-specific monoclonal antibody, 2A12, raised against ascidian DS (20).…”

“…7). Importantly, we have recently demonstrated that a unique iD (IdoUA(2-O-sulfate)-GalNAc(6-O-sulfate))-containing epitope is spatiotemporally expressed in particular regions of the developing mouse brain (20) and appears to be involved in the formation of neurites in cultured embryonic mouse * The work performed in Kobe was supported in part by HAITEKU (2004HAITEKU ( -2008) from the Japan Private School Promotion Foundation and Grants-in-Aid for Exploratory Research 17659020, Scientific Research-B 16390026, and Scientific Research on Priority Areas 14082207 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges.…”