[Show abstract][Hide abstract] ABSTRACT: Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33,
43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated
polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins.
Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin
nor hyaluronic acid. AS antibodies did not bind to heparan sulfate or heparin, but bound to 6-O-desulfated, N-desulfated and re-N-acetylated heparin to varying degrees. ACH55 bound to tri-desulfated and re-N-acetylated heparin but hardly bound to other modified heparins. NAH antibodies did not bind to heparin and modified heparins
but bound to heparan sulfate to varying degrees. NAH43 and NAH46 also bound to partially N-de-acetylated N-acetyl-heparosan. Immunohistochemical analysis in rat cerebella was performed with the antibodies. While NAH46 stained endothelia,
where heparan sulfate is typically present, neither ACH55 nor AS25 stained endothelia. On the contrary ACH55 and AS25 stained
the molecular layer of the rat cerebella. Furthermore, ACH55 specifically stained Purkinje cells. These results suggest that
there is unordinary expression of IdoA2S-GlcNAc and IdoA-GlcNAc in specific parts of the nervous system.
[Show abstract][Hide abstract] ABSTRACT: Both saturated and unsaturated forms of isomeric unsulfated glycosaminoglycan (GAG) oligosaccharides, i.e., tetrasaccharides of chondroitin (CH) and hyaluronan (HA), were analyzed by electrospray ionization mass spectrometry/mass spectrometry. Although the only structural difference between them was the hydroxyl group at the C-4 position in N-acetylhexosamine (GalNAc or GlcNAc, respectively), given the same m/z value of precursor ions, these isomers in both their saturated and unsaturated forms could be separated by careful examination of diagnostic fragment ions in their product ion mass spectra when the relative abundances of these fragment ions were considered. In addition, the product ion mass spectrum of the unsaturated HA tetrasaccharide was compared with its linkage isomer, N-acetylheparosan tetrasaccharide. In this case, the isomers were more easily differentiated by comparing their characteristic spectral patterns. By adopting this approach, systematic differentiation of isomeric unsulfated GAG oligosaccharides should be achieved by means of fragmentation. It should also contribute widely to GAG-related biochemical and medicinal research in the future.
Journal of the Mass Spectrometry Society of Japan 01/2007; 55(1):1-6. DOI:10.5702/massspec.55.1
[Show abstract][Hide abstract] ABSTRACT: In order to prepare a series of N-acetylheparosan (NAH)-related oligosaccharides, bacterial NAH produced in Escherichia coli strain K5 was partially depolymerized with heparitinase I into a mixture of even-numbered NAH oligosaccharides, having an unsaturated uronic acid (DeltaUA) at the non-reducing end. A mixture of odd-numbered oligosaccharides was derived by removing this DeltaUA in the aforementioned mixture by a 'trimming' reaction using mercury(II) acetate. Each oligosaccharide mixture was subjected to gel-filtration chromatography to generate a series of size-uniform NAH oligosaccharides of satisfactory purity (assessed by analytical anion-exchange HPLC), and their structures were identified by MALDITOF-MS, ESIMS, and 1H NMR analysis. As a result, a microscale preparation of a series of both even- and odd-numbered NAH oligosaccharides was achieved for the first time. The developed procedure is simple and systematic, and thus, should be valuable for providing not only research tools for heparin/heparan sulfate-specific enzymes and their binding proteins, but also precursor substrates with medical applications.
Carbohydrate Research 03/2006; 341(2):230-7. DOI:10.1016/j.carres.2005.11.013 · 1.93 Impact Factor