Program and Abstracts for the 2007 Meeting of the Society for Glycobiology

The fucoidan from Sargassum thunbergii was isolated by both hot water and enzyme hydrolyzing by taking the yield of extracted fucoidan, the content of sulfate radical and the content of polysaccharides as indicators. The orthogonal tests were designed to optimize the variable factors. The findings showed that the optimized factors for extracting fucoidan by hot water were temperature 98 , pH6, time 5h,the amount of 15 times water of sample added. The yield of fucoidan extracted was 3.05% and the content of sulfate radical was 0.87%. The optimized factors for enzyme hydrolyzing were composite enzymes added 1.7%, temperature 45 ,pH 5.5 and hydrolyzing time 60 minutes. The yield of fucoidan extracted was 3.6%,in which the content of sulfate radical was 1.1%. The extracted fucoidan by enzyme hydrolyzing was then purified by anion-exchange chromatography DEAE-52 and five fractions named F1, F2, F3, F4 and F5 were obtained, in which the content of sulfate radical were 0, 3.94%, 6.57%, 12.60%, 38.08% respectively, and the recoveries of polysaccharides were 1.58%, 4.45%, 4.08%, 16.15%, 6.83% respectively. F1 had no sulfate radical content and was not the constituent of fucoidan. F2、F3、F4、F5 were further purified by Sephadex G–200. Six peaks were appeared, which were named as f1、f2、f3、f4、f5、f6. f2 and f4 were probably coagulates of fucoidan. The molecular weights of other four fractions were 40.5 kDa, 71.3 kDa, 132.5 kDa, 79.9kDa respectively. (173) Conformational Analysis of a Dermatan Sulfate Derived Tetrasaccharide by NMR, Molecular Modeling and Residual Dipolar Coupling Alba Silipo; Zhenqing Zhang; F. Javier Cañada; Antonio Molinaro; Robert J. Linhardt; Jesùs Jimenez-Barbero Centro de Investigaciones Biològicas, CSIC, Madrid, Spain; Rensselaer Polytechnic Institute, Troy, NY, U.S.A.; University of Naples Federico II, Naples, Italy The solution conformation of a dermatan-derived tetrasaccharide: ΔHexA-(1-3)-GalNAc4S-β-(1-4)-IdoA-α-(1-3)-red-GalNAc4S (S is a sulfate group) has been explored by means of NMR spectroscopy, especially NOE-based conformational analysis. The tetrasaccharide was present as four species, two of them are chemically different by the anomeric orientation of the reducing 2deoxy-2-acetamido-galactose (red-GalNAc) residue, while the other two are originated by different conformations of the iduronic acid (IdoA) unit. The two α-β interconverting anomers were in a 0.6:1 ratio. Ring conformations have been defined by the analysis of 3JHH-coupling constants and inter-residual NOE contacts. Both 2-deoxy-2-acetamido-galactose residues (GalNAc) were found in the 4C1 chair conformation, the unsaturated uronic acid (ΔHex A) adopts a very major half-chair 1H2 conformation, while the IdoA residue stands either in the 1C4 chair or in the 2S0 skewed boat geometries, in a 4:1 ratio. There is a moderate flexibility of Φ and Ψ torsions as suggested by nuclear overhauser effect (NOE), molecular modeling (MM) and molecular dynamics (MD) studies. This fact has been further investigated by residual dipolar couplings (RDCs). One bond C-H RDCs (1DCH) and long range H-H (3DHH) RDCs were measured for the tetrasaccharide in a phage solution and interpreted in combination with restrained MD simulation. The RDC-derived data substantially confirmed the validity of the conformer distribution resulting from the NOEderived simulations, but allowed an improved definition of the conformational behavior of the oligosaccharides in solution. In summary, the data showed a moderate flexibility of the four tetrasaccharide species at the central glycosidic linkage. (174) An LC/MS Platform for Glycomics of Glycosaminoglycans in Mammalian Organ Tissues Joseph Zaia; Xiaofeng Shi Boston University School of Medicine, Boston, MA Glycosaminoglycans (GAGs) are linear sugar polymers covalently bound to a core protein. They are abundant on the cell surface and in the extracellular matrix, serving a wide range of functions. The structure of GAGs exhibits a great degree of polydispersity in composition, chain length, sulfation, acetylation and epimerization pattern. Glycomic studies of GAGs in different tissues of different species aim to understand how the structures and quantities of GAGs are related to aging, development, mutation and specific diseases. Mass spectrometry is a powerful tool for structural analysis of GAGs. Its use, however, depends on the availability of a general method for extraction of GAGs from tissue. We developed such a glycomic platform consisting of extracting GAGs from various rat tissues, and subjecting the GAGs to on-line LC/MS and LC/MS/MS analysis. The extraction consists of sequential chondroitin and heparin sulfate lyase digestion and weak Annual Conference of the Society for Glycobiology Conference Abstracts 1249 ion (DEAE) exchange workup. The LC/MS utilizes amide-80 as chromatography and ESI-QIT as mass detector. We have demonstrated that LC/MS and LC/MS/MS analysis of exhaustive digested of both chondroitin sulfate (CS) and heparan sulfate (HS) from these tissues give detailed information of the disaccharide composition of these GAGs and their differences among different tissues. An improved capillary electrophoresis method was also developed to serve as an orthogonal approach for disaccharide analysis and quantification. This platform will be useful for glycomic studies of GAGs in tissues directly harvested from mammals. (175) Tissue – Based Glycomics using Amide-HILIC LCTandem MS Alicia M. Hitchcock; Catherine E. Costello; Joseph Zaia Boston University School of Medicine, Boston, MA Homeostasis of connective tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans and glycosaminoglycans (GAGs). To define the functional roles of glycan expression during disease formation it is necessary to determine their patterns of expression as a function of tissue location. Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. This analysis enables simultaneous profiling of the expression of saturated non-reducing end, linker region, and delta-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results generate an unparalleled level of detail in profiling structural changes to connective tissue-derived GAGs during disease and development. Sulfated GAGs were extracted from papain-digested connective tissue samples using a streamlined multi-step procedure. Oligosaccharides were partially depolymerized followed by derivatization with 2-anthranilic-3,4,5,6-d4 acid. Derivatized tissue samples were cleaned, spiked with standard CSA-d0-2AA, and subjected to an online amide-HILIC-LC-MS/MS platform using an Esquire 3000 ion trap. A combination of MS and tandem MS analysis was employed for detailed structural characterization. Longer oligosaccharides may contain differing domains of sulfation and epimerization that may play crucial roles in development and disease processes. Characterization of the actual sulfation and epimerization positions within differing domains is also investigated using a high resolution, high sensitivity Orbitrap