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

Abstract

Mammalian brains contain significant amounts of chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid chains. CS/DS chains isolated from embryonic pig brains (E-CS/DS) promote the outgrowth of neurites in embryonic mouse hippocampal neurons in culture by interacting with pleiotrophin (PTN), a heparin-binding growth factor. Here, we analyzed oligosaccharides isolated from E-CS/DS, which showed that octasaccharides were the minimal size capable of interacting with PTN at a physiological salt concentration. Five and eight sequences were purified from fluorescently labeled PTN-bound and -unbound octasaccharide fractions, respectively, by enzymatic digestion followed by PTN-affinity chromatography. Their sequences were determined by enzymatic digestion in conjunction with high performance liquid chromatography, revealing a critical role for oversulfated D and/or iD disaccharides in the low yet significant affinity for PTN, which is required for neuritogenesis. The critical D and iD units are GlcUA(2-O-sulfate)␤1-3GalNAc(6-O-sulfate) and IdoUA(2-O-sulfate)␣1-3GalNAc(6-O-sulfate), respectively, where IdoUA represents L-iduronic acid. In contrast, high affinity interactions with PTN required decasaccharides with E units (GlcUA␤1-3GalNAc(4, 6-O-disulfate)), B units (GlcUA(2-O-sulfate)␤1-3GalNAc(4-O-sulfate)), and/or their IdoUA-containing counterparts (iE and iB) in addition to D/iD units, although the biological significance of such strong interactions remains to be investigated. Thus, chain size and composition are crucial to the interaction with PTN, and PTN binds to multiple sequences in E-CS/DS chains with distinct affinity. Notably, not only heparan sulfate but also CS/DS hybrid chain structures of mammalian brains contain a high degree of microheterogeneity with a cluster of oversulfated disaccharides and appear to play roles in regulating the functions of PTN.Chondroitin sulfate (CS) 4 and dermatan sulfate (DS), a class of glycosaminoglycan (GAG)-type polysaccharides, occur covalently attached to proteoglycan (PG) core proteins (1). Like heparan sulfate (HS), another class of GAGs, CS/DS chains are abundant at cell surfaces and in the extracellular matrices. They regulate cell division, adhesion, and morphogenesis through direct binding to secreted signaling proteins, thereby modulating their activities, or through interactions with extracellular matrix molecules (2-6, for reviews, see a Ref. 7). CS and DS typically have backbones consisting of repeating disaccharide units of -GlcUA-GalNAc-and -IdoUA-GalNAc-, respectively, where IdoUA represents L-iduronic acid. Notably, hybrid chains, composed of both units in varying proportions, also exist (8). These 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. These structural characteristics are strictly regulated, as revealed by the compositional analysis of CS/DS chai...

Biosynthesis of Dermatan Sulfate

“…AH-Sepharose, Sephadex G-25, Red-Sepharose, and ConA-Sepharose gels, Mono-Q 5/5, Superose 12 HR 10/30, Mono-Q PC 1.6/5 (operated with Smart System), Superdex Peptide 10/300 GL, and PD-10 columns were obtained from Amersham Biosciences, as was D- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]glucose (57 mCi/ mmol). D- [5-3 H]Glucose (20 Ci/mmol) was from PerkinElmer Life Sciences, and inorganic carrier-free […”

“…Enzyme samples, containing 3 mg of BSA as carrier unless otherwise stated, were desalted at 4°C on Sephadex G-25 columns (0.7 ϫ 3 cm), equilibrated with desalting buffer (20 mM MES (pH 5.5 at 37°C), 10% glycerol, 0.5 mM EDTA, 0.1% Triton X-100, 1 mM DTT, protease inhibitors). Incubations were performed in a 100-l final volume of 0.8ϫ desalting buffer, 0.5 mg of BSA, 2 mM MnCl 2 , 0.5% Nonidet P-40, and 30,000 dpm H]dK4 or [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]dK4 (ϳ200 M HexA for both substrates). Incubations were done at 37°C for 2-14 h and stopped by boiling for 5 min, and samples were centrifuged.…”

Conformational Studies on Five Octasaccharides Isolated from Chondroitin Sulfate Using NMR Spectroscopy and Molecular Modeling

“…More recently, octasaccharide sequences, recognized by a heparin-binding growth factor pleiotrophin, were isolated from the CS/DS hybrid chains of embryonic pig brains. They were derived from the corresponding nonasaccharide sequences, which contained A-D, iA-D, C-D, or iC-D tetrasaccharide, and were flanked by two IdoA residues (37). In view of the data obtained from the present study, the above-described evidence may suggest a contribution of the IdoA residue(s) in the vicinity of the A-D sequence to the concentration of the negative charges in the center of the 473HD epitope sequences, providing additional specificity.…”

Structure of Pleiotrophin- and Hepatocyte Growth Factor-binding Sulfated Hexasaccharide Determined by Biochemical and Computational Approaches

“…In this study, a novel PTN-binding hexasaccharide was isolated from the SS-CS/DS hybrid chains after digestion with chondroitinase AC-I and its strong binding with HGF was also demonstrated, suggesting that the PTN-and HGF-binding domains in SS-CS/DS overlap partially, if not completely. Intriguingly, the isolated hexasaccharide differed in disaccharide composition and sequence from the PTN-binding octasaccharides isolated from E-CS/DS (21), which prompted us to investigate the three-dimensional structure and electrostatic potential (ESP) distribution of the PTN-binding and non-binding sequences using methods previously reported for comparing multiple CS oligosaccharides recognized by monoclonal antibodies (23,24). Computational chemistry using molecular modeling, simulation and ESP calculations revealed the three-dimensional structure and distribution of ESP on the molecular surface not only for the oligosaccharides but also for PTN, which allowed us to predict the binding pocket of the PTN molecule and gain new insights into the molecular mechanism by which the oligosaccharides are recognized by PTN.…”

“…A series of PTN-binding octasaccharides with unique structures have been isolated from embryonic pig brain (E-CS/DS). These were prepared by digestion with CSase B specific to IdoUA (21), suggesting that the isolated sequences, which lacked IdoUA, were flanked by two IdoUA residues within the CS/DS hybrid chains. Thus, the identification of unique PTN/HGF-binding sequences is likely to explain the presence of similar functional activity in the apparently structurally different E-CS/DS and SL-CS/DS chains.…”