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Analysis of Mucin-Type O-Linked Oligosaccharides 19119116Structural Analysisof Mucin-Type O-Linked OligosaccharidesAndré Klein, Gérard Strecker, Geneviève Lamblin,and Philippe Roussel1. IntroductionThe carbohydrate moiety of mucin is characterized by the presence of oligosaccha-rides linked to the peptide backbone by an O-glycosidic linkage between an N-acetylgalactosamine residue and a hydroxylated amino acid (serine or threonine).These linkages are alkali labile and the carbohydrate chains can be released as oli-gosaccharide-alditols by a β-elimination, with NaOH in the presence of NaBH4. Thestructures of carbohydrate chains found in mucins can be as simple as the disaccharideNeuAc α2→6GalNAc in ovine submaxillary mucin and as complex as the ones foundin human respiratory or salivary mucins, in which several hundred different carbohy-drate chains exist (1,2). This diversity is generated (1) by the different monosaccha-rides constituting the glycans, generally fucose, galactose, N-acetylgalactosamine,N-acetylglucosamine, and N-acetyl neuraminic acid, but also other monosaccharidessuch as ketodesoxynonulosonic acid or N-glycolylneuraminic acid, and, finally theoccurance of sulfation of galactose and N-acetylglucosamine (3,4); and (2) by the dif-ference in length, in branching, and by the occurrence of all the different possiblelinkages between the constituting monosaccharides. The diversity of mucin-type oli-gosaccharides can be extreme. For example, 88 oligosaccharides have been isolatedfrom the respiratory mucins of a single individual (5–10) and more than 150 have beenisolated from the jelly coat from eggs of different species of amphibians (11–15).This chapter gives an overall idea of the strategy of elucidation of primary structureof glycans, the most current nuclear magnetic resonance (NMR) techniques used instructure determination, and some of the mass spectrometry (MS) techniques avail-able for the glycobiologist.1.1. StrategyThe amount of pure oligosaccharide (or of a mixture of two compounds, or three atthe most) and the facilities that are available in the laboratory environment will defineFrom:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 192 Klein et al.the strategy. Two types of techniques are used: a destructive technique, MS and anondestructive technique, NMR. Figure 1 summarizes the sequence of techniques.When the amount of oligosaccharide permits the use of NMR first, especially if two-dimentional (2D) NMR can be performed, MS is only used to confirm the structure ifit is a novel one; in most cases, it is not needed. MS is quite useful when there is notenough material for NMR or when the mixture studied is too complex.1.2. Nuclear Magnetic ResonanceNMR spectroscopy constitutes the most suitable method for the structure determi-nation of carbohydrate chains. This method was introduced in the 1970s and rapidlyreceived a large application for analyzing the sequence of N-acetyllactosamine- andoligomannosidic-type glycans.Originally, and before the development of 2D NMR spectroscopy, the method waslimited to one-dimentional 1H-NMR spectroscopy, which has led to the concept of“structural-reporter groups.” In these conditions, depending on the field of spectrom-eter (600–300 MHz), 20–100 nmol will constitute a sufficient amount of material toapply a “finger-print” method. Nevertheless, the method is restricted to compoundswhich are members of a series of closely related sequences, as it is happily the case formost of O-glycans.When the material is available in the range of 0.1–5 µmol, the de novo structuralelucidation of the sequences can be easily deduced from the compilation of data fur-nished by various homo and heteronuclear 2D NMR methods.1.3. Mass SpectrometryMS has become an indispensable tool for the determination of carbohydrate struc-tures. The information provided by this methodology ranges from the accurate mo-lecular weight determination to the complete primary structure with a sensitivity suchthat only picomoles of oligosaccharides are necessary. These remarkable advanceshave been made possible with the appearence of novel methods of ionization such asfast atom bombardment ionization (FAB), electrospray ionization (ESI), and matrix-assisted laser desorption ionization (MALDI).2. Methods2.1. Nuclear Magetic Resonance2.1.1. Proton-NMR as a Fingerprinting MethodThe proton-NMR method was developed by Vliegenthart and colleagues during the1970s and essentially applied to the structure determination of N-glycans of the N-acetyllactosamine and oligomannoside type (16). More recently, a similar procedurewas summarized for the primary structural analysis of oligosaccharide-alditol releasedfrom mucin-type O-glycosylproteins (17). This method is based on the recognition ofsome atom resonances that constitute probes for representative structural elements.These structural-reporter groups resonate outside the bulk constituted by thenonanomeric protons. 1H-NMR structural-reporter group signals correspond to thefollowing atom resonances: anomeric protons; GalNAc-ol H-2, H-4, H-5 and H-6'Fig. 1 Analysis of Mucin-Type O-Linked Oligosaccharides 193atoms; Gal H-3 and H-4 atoms; Fuc H-5 and H-6 atoms; NeuAc H-3ax and H-3eqatoms; and CH3 of the acetamido groups.The first step of spectrum analysis consists of the identification of the core region(Table 1), based on the characteristic chemical shifts of the H-2 and H-5 atom reso-nances of the GalNAc-ol unit. Moreover, the quadruplet of the H-6' signal of GalNAc-ol is upfield shifted out of the bulk at δ ~ 3.50 ppm in the case of an O-6 substitutionwith sialic acid. The presence of α-2,3- or α-2,6-linked sialic acid is clearly shown bythe respective chemical shift of the H-3ax and H-3eq signals of the monosaccharides.The H-3ax and H-3eq resonances of the α-2,3-linked NeuAc are systematicallydownfield shifted, compared to the corresponding signals of α-2,6-linked NeuAc.The attachment of NeuAc at O-3 of a Gal unit causes downfield shifts of the Galstructural-reporter groups, as clearly indicated in Fig. 2, in which the NMR spectra ofasialo and sialo glycans are compared (compounds N-1 and A-1).Fig. 1. Strategy of elucidation of oligosaccharide primary structure. 194Klein et al.194Table 1Chemical Shifts of GalNAc-ol Residues Characteristic of the Nature of Oligosaccharide-Alditol CoresGal(β1–3)GalNAc-ol GlcNAc(β1–3)GalNAc-ol Gal(β1–3)[GlcNAc(β1–6)]GalNAc-ol NeuAc(α2–6)GalNAc-olH-2 4.393 4.286 4.391 4.246H-3 4.063 3.995 4.069 3.846H-4 3.506 3.546 3.468 3.411H-5 4.193 4.141 4.277 4.020H-6' 3.628 ND ND 3.532Gal(β1–3)[NeuAc(α2–6)]GalNAc-ol GlcNAc(β1–3)[NeuAc(α2–6)]GalNAc-ol GlcNAc(β1–6)GalNAc-olH-2 4.378 4.260 4.242H-3 4.055 3.984 3.841H-4 3.534 ND 3.379H-5 4.244 4.185 4.021H-6' 3.486 3.490 3.933 Analysis of Mucin-Type O-Linked Oligosaccharides 195Fig. 2. 1H-NMR spectra of three oligosaccharide-alditols. N-1, basic structure devoid offucose and sialic acid residue; N-2, downfield shift of Gal3 H-1 owing to the α-1,2 fucose; A-1, downfield shift of Gal3H-1 and H-3 owing to the α-2,3 sialylation. The first superscript afterthe abbreviated name of a monosaccharide residue indicates to which position of the adjacentmonosaccharide it is glycosidically linked (e.g., Gal4 in the case of Galβ1→4 GlcNAcβ1→).Fucose units can be easily identified according to the presence of methyl reso-nances at ~ 1.2 ppm. The α-1,2 (H), α-1,3 (Lex), and α-1,4 (Lea) linkages are deducedfrom the position of H-1, H-5, and H-6 resonances. For instance, the Lexand Leaepitopes can be characterized on the basis of their H-1 (Lex: δ ~ 5.13–5.14; Lea: δ ~5.02–5.05), and H5 (Lex: δ ~ 4.80–4.85; Lea: δ ~ 4.86–4.88) resonances, whereas the 196 Klein et al.H-5 signal of α-1,2-linked Fuc is observed at δ ~ 4.25–4.34 ppm. These NMR dataimply, respectively, the presence of type 2 (Galβ1-4GlcNAc) and type 1 (Galβ1-3GlcNAc) backbone structures.The analysis of compounds with a higher complexity in backbone sequence cannotbe developed in some pages, and such analyses often call for additional chemical (me-thylation analysis) or physical (MS, nuclear Overhauser effect [NOE] measurements)operations.Figures 3 and 4 give examples of complex backbones. Compounds were analyzedby methylation analysis, which precises the location of the hydroxyl groups impli-cated in the glycosidic linkages. The comparison of the spectra N-4B, N-4A, N-6B,and N-8 clearly indicates the presence of the same sequence Gal(β1-4)GlcNAc(β1-6)GalNAc-ol, as shown by the NMR parameters of Gal4and GlcNAc6. This comparisonbetween N-1, A-1, and A-3 (Fig. 2) also gives the chemical shift of the sialylated andterminal Gal unit O-3 linked to GalNAc-ol. Consequently, the upper branch of com-pound A-3 is composed of two Gal and two GlcNAc units. Other models (not shownhere) also possess the sequences Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)GlcNAc orGal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc, with additional Fuc or NeuAc attached tothe terminal galactose units. Since the presence of these peripheral monosaccharidesaffects essentially the chemical shifts of the terminal galactose, the anomeric protonscan be assigned step-by-step.The compilation of 168 NMR spectra which are included in the review of Kamerlinget al. (17) clearly shows that a spectrum is unique and can be used as an “identitycard.” This review presents the carbohydrate chains in a logical order and gives aclassification according to the nature of the core, the nature of the backbone, and theperipherical monosaccharides. Actually, the nature of the backbone can be often di-Fig. 2. Continued. Analysis of Mucin-Type O-Linked Oligosaccharides 197rectly deduced from the linkage of the fucose units, as discussed previously. When thepresence of these structural elements has been established, the number of possibilitiesdecreases deeply, and a survey of the corresponding class of oligosaccharide-alditolsfurnishes rapidly the structure of the compound. Most of the O-glycans that constitutethe carbohydrate moiety of mucins isolated from human tissues have now been de-scribed, but completely new structures can be isolated from other biological sources.If a sufficient amount of material is available, de novo structural elucidation of glycansequences should be performed by 2D NMR spectroscopy.Fig. 3. 1H-NMR spectra of oligosaccharide-alditols with the common tetrasaccharide N-1(see Fig. 2) variously substituted. 198 Klein et al.2.1.2.De novo Structural Elucidation of Glycan Sequencesby NMR SpectroscopyHomonuclear correlated spectroscopy (COSY) provides information on directlycoupled protons with regard to coupling constants. Starting from the anomeric proton,the H-2, H-3, H-4, and so on, atom resonances, which were masked in the bulk, can beassigned. Nevertheless, such an assignment is generally difficult when the cross peaksFig. 3. Continued.Fig. 4. 1H-NMR spectrum of oligosaccharide-alditols with the common tetrasaccharide N-1(see Fig. 2) variously substituted. Analysis of Mucin-Type O-Linked Oligosaccharides 199occur around the diagonal. Two-dimensional total correlation spectroscopy (2D 1H-TOCSY) can be used to characterize all the proton resonances. A transfer of magneti-zation from H-1 to H-6 is observed in the case of the α− and β-gluco configuration(J1,2–J4,5 ~ 8 Hz), wheareas the small J4,5 that characterizes the galacto configurationinterrupt the assignment at the H-4 resonance. Relayed COSY spectra have the advan-tage of successively assigning the H-2 (COSY), H-3 (one-step-relayed COSY), and H-4 (two-steps-relayed COSY) atom resonance of the carbohydrate units.The example given in Fig. 5 clearly shows the presence of two β-Gal, one α-Galand one GlcNAc units. For N-acetylglucosamine, H-2, H-3, and H-4 signals are trip-Fig. 4. Continued. 200Klein et al.Fig. 5. COSY (left) and HMQC (right) NMR spectra of an heptasaccharide-alditol isolated from the oviducal mucin of Xenopus laevis. 2II,proton H-2 of monosaccharide unit II. For the COSY spectrum, starting from the anomeric proton (i.e., 1III) the protons 2III, 3III, and 4IIIare [...]... structure of neutral and acidic oligosaccharide-alditols derived from the jelly coat of the Mexican axolotl. Occurence of oligosaccharide with fucosyl α( 1-3 ) fucosyl (α 1-4 )-3 deoxy-D-glycero-D-galacto-nonulosonic acid and galac- tosyl (α 1-4 ) [fucosyl(α 1-2 )]galactosyl(β 1-4 )N-acetylglucosamine sequence. Eur. J. Biochem. 207, 995–1002. 12. Strecker, G., Wieruszeski, J M., Fontaine, M. D., and Plancke, Y. (1994)... Gal(β 1-4 )GlcNAc(β 1-6 ) GalNAc-ol, as shown by the NMR parameters of Gal 4 and GlcNAc 6 . This comparison between N-1, A-1, and A-3 (Fig. 2) also gives the chemical shift of the sialylated and terminal Gal unit O-3 linked to GalNAc-ol. Consequently, the upper branch of com- pound A-3 is composed of two Gal and two GlcNAc units. Other models (not shown here) also possess the sequences Gal(β 1-3 )GlcNAc(β 1-3 )Gal(β 1-4 )GlcNAc... the case of an O-6 substitution with sialic acid. The presence of -2 , 3- or -2 ,6-linked sialic acid is clearly shown by the respective chemical shift of the H-3ax and H-3eq signals of the monosaccharides. The H-3ax and H-3eq resonances of the -2 ,3-linked NeuAc are systematically downfield shifted, compared to the corresponding signals of -2 ,6-linked NeuAc. The attachment of NeuAc at O-3 of a Gal unit... A. Corfield © Humana Press Inc., Totowa, NJ Analysis of Mucin-Type O -Linked Oligosaccharides 195 Fig. 2. 1 H-NMR spectra of three oligosaccharide-alditols. N-1, basic structure devoid of fucose and sialic acid residue; N-2, downfield shift of Gal 3 H-1 owing to the -1 ,2 fucose; A- 1, downfield shift of Gal 3 H-1 and H-3 owing to the -2 ,3 sialylation. The first superscript after the abbreviated... methyl reso- nances at ~ 1.2 ppm. The -1 ,2 (H), -1 ,3 (Le x ), and -1 ,4 (Le a ) linkages are deduced from the position of H-1, H-5, and H-6 resonances. For instance, the Le x and Le a epitopes can be characterized on the basis of their H-1 (Le x : δ ~ 5.13–5.14; Le a : δ ~ 5.02–5.05), and H5 (Le x : δ ~ 4.80–4.85; Le a : δ ~ 4.86–4.88) resonances, whereas the Analysis of Mucin-Type O -Linked Oligosaccharides... possesses an - galacto configuration, is easily distinguished from α-galactose owing to the overlap of the correlations H-6→H-5→H-4 and H-1→H-2→H-3→H-4. The heteronuclear multiple quantum-coherence spectroscopy (HMQC) relies on the 1 H and 13 C, which are directly attached. The position of the glycosidic linkage is clearly observed owing to a strong downfield shift (4–10 ppm) that affect the substi- tuted... II, and IV possess, respectively the β-Gal, β-Glc, and α-Gal configuration. Hexose and N- acetylhexosamine are discriminated according to their H-2 resonances, strongly deshielded in the case of N-acetylhexosamine. For the HMQC spectrum, a comparison with the correspond- ing COSY spectrum allows the assignment of the 13 C resonances (i.e. 4 III represents the corre- lation peak between Gal III 1 H-4/ 13 C-4).... different monosaccha- rides constituting the glycans, generally fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetyl neuraminic acid, but also other monosaccharides such as ketodesoxynonulosonic acid or N-glycolylneuraminic acid, and, finally the occurance of sulfation of galactose and N-acetylglucosamine (3,4); and (2) by the dif- ference in length, in branching, and by the occurrence... oligosaccharide and confirm the assignment of each anomeric proton. 194Klein et al. 194 Table 1 Chemical Shifts of GalNAc-ol Residues Characteristic of the Nature of Oligosaccharide-Alditol Cores Gal(β1–3)GalNAc-ol GlcNAc(β1–3)GalNAc-ol Gal(β1–3)[GlcNAc(β1–6)]GalNAc-ol NeuAc(α2–6)GalNAc-ol H-2 4.393 4.286 4.391 4.246 H-3 4.063 3.995 4.069 3.846 H-4 3.506 3.546 3.468 3.411 H-5 4.193 4.141 4.277 4.020 H-6'... Gal H-3 and H-4 atoms; Fuc H-5 and H-6 atoms; NeuAc H-3ax and H-3eq atoms; and CH 3 of the acetamido groups. The first step of spectrum analysis consists of the identification of the core region (Table 1), based on the characteristic chemical shifts of the H-2 and H-5 atom reso- nances of the GalNAc-ol unit. Moreover, the quadruplet of the H-6' signal of GalNAc- ol is upfield shifted out of the . GalNAc-ol H-2, H-4, H-5 and H-6'Fig. 1 Analysis of Mucin-Type O-Linked Oligosaccharides 193atoms; Gal H-3 and H-4 atoms; Fuc H-5 and H-6 atoms; NeuAc H-3ax. withfucosyl α( 1-3 ) fucosyl (α 1-4 )-3 deoxy-D-glycero-D-galacto-nonulosonic acid and galac-tosyl (α 1-4 ) [fucosyl(α 1-2 )]galactosyl(β 1-4 )N-acetylglucosamine sequence.