NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-ß-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-ß-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-ß-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the ß-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

A glycosaminoglycan microarray identifies the binding of SARS-CoV-2 spike protein to chondroitin sulfate E.

Heparan sulfate (HS), a sulfated glycosaminoglycan (GAG), was reported to be a necessary host attachment factor that promotes SARS-CoV-2 infection. In this study, we developed GAG microarrays based on fluorescence detection for high-sensitivity screening of the GAG-binding specificity of proteins and applied it for the analysis of SARS-CoV-2 spike (S) protein. Among the 20 distinct GAGs, the S protein bound not only to heparin (HEP)/HS but also to chondroitin sulfate E (CSE) in a concentration-dependent manner. We then analyzed the specificity of each subunit of the S protein. While the S1 subunit showed exclusive binding to HEP, the S2 subunit also bound to CSE and HEP/HS. CSE might act as an alternative attachment factor for HS in SARS-CoV-2 infection.

Evaluation of intermolecular association of glycosaminoglycan oligosaccharides using nanoelectrospray ionization mass spectrometry.

This study examines the non-covalent interactions between glycosaminoglycan (GAG) oligosaccharides using nanoelectrospray ionization mass spectrometry (nanoESI-MS). It is the first time that interactions between oligosaccharides have been observed using MS. The importance of interactions between GAGs has recently attracted much interest because they are related to biological functions. For instance, hyaluronic acid (HA) is known to associate with chondroitin sulfates (CSs), although the details of the interaction remain unclear. In general, non-covalent interactions between glycans are too weak to detect by general means. In this work, we applied nanoESI-MS with high sensitivity, which is widely used to observe non-covalent interactions, to investigate the interaction between HA and CSs. HA and CS oligosaccharides are used to discuss the results in a simplified manner. Our approach is aimed at interpreting the behavior of GAG polysaccharides from the information obtained using the oligosaccharides. HA and CS tetrasaccharides were demonstrated to associate to form heterodimer ions that were easily detected using nanoESI-MS. We also determined the stoichiometry of the interaction and calculated the K(d) values of the interactions between HA and CS tetrasaccharides. How these structures affect the strength and stability of the non-covalent complexes is discussed. Further study of the interactions between HA and CS oligosaccharides will clarify the biological meaning of the coexistence of HA and CS in body fluids and tissues.

The Galß-(syn)-gauche configuration is required for galectin-recognition disaccharides.

BACKGROUND: Galectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant (K(d)). METHODS: In the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1-9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families. RESULTS: An empirical [Galß-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, "Galß1-4-L-Fuc" [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to 'axial' 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration "Galß-(syn)-gauche". The rule also worked perfectly for differentiation of galectins from other types of lectins. GENERAL SIGNIFICANCE: The present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands.

Desulfated galactosaminoglycans are potential ligands for galectins: evidence from frontal affinity chromatography.

Galectins, a group of beta-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of betaGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants.

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.

Multistage mass spectrometric sequencing of keratan sulfate-related oligosaccharides.

To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-MSn fragmentation experiments were carried out for 10 KS-related oligosaccharides of defined structure. Under the experimental conditions employed, fully charged molecular-related ions were observed as dominant peaks in all MS(1) spectra, which clearly reflected the number of sulfates and sialic acids in the oligosaccharide structures. In the subsequent MS2, almost all of the oligosaccharides gave fragment ions corresponding to their dehydrated molecular-related ions as well as (0,2)A(r) scission ions (according to the nomenclature developed by Domon and Costello, where "r" represents the reducing end in this study). Further fragmentation of the (0,2)A(r) ions in MS3 predominantly yielded the corresponding (2,4)A(r) ions. Finally, in MS(4), these (2,4)A(r) ions were subjected to extensive glycosidic cleavage. Hence, the MS4 data of KS oligosaccharides provided sufficient information for their sequence determination. In addition, some important features of MSn fragmentation became evident. These findings should lead to the establishment of consensus rules applied for KS oligosaccharides, including those previously unidentified, and also accelerate functional studies on KS, i.e., KS-related glycosaminoglycomics.

Systematic identification of N-acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation.

Recently, a useful procedure for the preparation of both even- and odd-numbered series of N-acetylheparosan (NAH) oligosaccharides was established. The present report describes findings when these NAH oligosaccharides were subjected to comparative mass spectrometry (MS)/MS fragmentation analysis by matrix-assisted laser desorption/ionization (MALDI)-LIFT-time-of-flight (TOF)/TOF-MS/MS, and electrospray ionization (ESI) collision-induced dissociation (CID) MS/MS. The resultant fragment ions were systematically assigned to elucidate fragmentation characteristics. In the MALDI-LIFT-MS/MS experiments, all the NAH oligosaccharides underwent unique glycosidic cleavages that included B-Y ion cleavages (nomenclature system of Domon and Costello, Glycoconjugate J. 1988; 5: 397) at the C-1 side, and C-Z ion cleavages at the C-4 side, with respect to glucuronic acid (GlcA). In addition, (0,2)A and/or (0,2)X cross-ring cleavages were observed for relatively small oligosaccharides. The former observation clearly reflects the occurrence of a GlcA-N-acetylglucosamine (GlcNAc) alternating structure of NAH, while the latter feature implies the occurrence of the -beta-1-4-glucuronide linkage. Extensive glycosidic cleavages were also observed in the ESI-CID-MS/MS fragmentation, though cleavage specificity was less evident than in the case of MALDI-LIFT-TOF/TOF-MS/MS. The information obtained in this study should be valuable for understanding both biosynthetic and degradation processes of NAH and its derivatives including heparin and heparan sulfate, as well as artificially modified NAH oligosaccharides.

Microscale preparation of even- and odd-numbered N-acetylheparosan oligosaccharides.

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.

Fragmentations of isomeric sulfated monosaccharides using electrospray ion trap mass spectrometry.

Electrospray ionization combined with ion trap mass spectrometry (ESI-ITMS) is a powerful tool for structural analysis of complex carbohydrates. Although its application to sulfated glycans has been limited so far, it should provide critical information, such as sulfate positions, on their structures. In this work, MS(n) spectra of nine monosulfated monosaccharides, consisting of five hexoses and four N-acetylhexosamines, were measured in negative ion mode to find basic fragmentation rules for sulfated sugars. Two pairs of positional isomers with respect to sulfation, i.e., Gal4S and Gal6S, and GalNAc4S and GalNAc6S, showed characteristic fragmentation patterns in MS(3), and could be discriminated from one another by the appearance of particular diagnostic fragment ions that characterize individual isomers. It was also demonstrated that, even if a mixture of these positional isomers was analyzed, the proportion of each species could be estimated through analysis of the abundance ratios of the diagnostic ions. However, 3-O-sulfated saccharides (Glc3S and GlcNAc3S) gave a single abundant diagnostic ion in MS(2) corresponding to the hydrogensulfate ion, [OSO(3)H](-), and this characteristic clearly differentiated them from their positional isomers. In contrast, 6-O-sulfated diastereomers consisting of two groups, Glc6S, Man6S, Gal6S, and GlcNAc6S, GalNAc6S, could not be discriminated by the types of fragment ions; however, the abundance ratios of particular fragment ions differed significantly between Glc(NAc)6S and Gal(NAc)6S. Since ESI-ITMS yielded large quantities of useful information on structures of monosulfated hexoses and N-acetylhexosamines in an extremely simple and reproducible manner, establishment of a comprehensive strategy based on ESI-ITMS(n) appears to be a promising technique for structural elucidation of sulfated complex carbohydrates.