Glycoprotein methods protocols - biotechnology 048-9-191-209.pdf

Glycoprotein methods protocols - biotechnology

Analysis of Mucin-Type O-Linked Oligosaccharides 191

191

16

Structural Analysis

of Mucin-Type O-Linked Oligosaccharides

André Klein, Gérard Strecker, Geneviève Lamblin,

and Philippe Roussel

1 Introduction

The 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 The structures of carbohydrate chains found in mucins can be as simple as the disaccharide NeuAcα2→6GalNAc in ovine submaxillary mucin and as complex as the ones found

in 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 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 of all the different possible linkages between the constituting monosaccharides The diversity of mucin-type oli-gosaccharides can be extreme For example, 88 olioli-gosaccharides have been isolated

from the respiratory mucins of a single individual (5–10) and more than 150 have been isolated 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 structure

of glycans, the most current nuclear magnetic resonance (NMR) techniques used in structure determination, and some of the mass spectrometry (MS) techniques avail-able for the glycobiologist

1.1 Strategy

The amount of pure oligosaccharide (or of a mixture of two compounds, or three at the most) and the facilities that are available in the laboratory environment will define

From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

enough material for NMR or when the mixture studied is too complex.

1.2 Nuclear Magnetic Resonance

NMR spectroscopy constitutes the most suitable method for the structure determi-nation of carbohydrate chains This method was introduced in the 1970s and rapidly

received a large application for analyzing the sequence of N-acetyllactosamine- and

oligomannosidic-type glycans

Originally, and before the development of 2D NMR spectroscopy, the method was limited 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 to apply a “finger-print” method Nevertheless, the method is restricted to compounds which are members of a series of closely related sequences, as it is happily the case for

most of O-glycans.

When the material is available in the range of 0.1–5 µmol, the de novo structural

elucidation 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 Spectrometry

MS 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 such that only picomoles of oligosaccharides are necessary These remarkable advances have been made possible with the appearence of novel methods of ionization such as fast atom bombardment ionization (FAB), electrospray ionization (ESI), and matrix-assisted laser desorption ionization (MALDI)

2 Methods

2.1 Nuclear Magetic Resonance

2.1.1 Proton-NMR as a Fingerprinting Method

The proton-NMR method was developed by Vliegenthart and colleagues during the

1970s and essentially applied to the structure determination of glycans of the

N-acetyllactosamine and oligomannoside type (16) More recently, a similar procedure

was summarized for the primary structural analysis of oligosaccharide-alditol released

from mucin-type O-glycosylproteins (17) This method is based on the recognition of

some atom resonances that constitute probes for representative structural elements These structural-reporter groups resonate outside the bulk constituted by the nonanomeric protons 1H-NMR structural-reporter group signals correspond to the following atom resonances: anomeric protons; GalNAc-ol H-2, H-4, H-5 and H-6'

Analysis of Mucin-Type O-Linked Oligosaccharides 193

atoms; Gal H-3 and H-4 atoms; Fuc H-5 and H-6 atoms; NeuAc H-3ax and H-3eq atoms; 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 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 substitution with sialic acid The presence of α-2,3- or α-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 α-2,3-linked NeuAc are systematically downfield 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 Gal

structural-reporter groups, as clearly indicated in Fig 2, in which the NMR spectra of

asialo and sialo glycans are compared (compounds N-1 and A-1)

Fig 1 Strategy of elucidation of oligosaccharide primary structure.

Klein et al.

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

Gal(β1–3)[NeuAc(α2–6)]GalNAc-ol GlcNAc(β1–3)[NeuAc(α2–6)]GalNAc-ol GlcNAc(β1–6)GalNAc-ol

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 α-1,2 fucose;

A-1, downfield shift of Gal 3 H-1 and H-3 owing to the α-2,3 sialylation The first superscript after the abbreviated name of a monosaccharide residue indicates to which position of the adjacent monosaccharide it is glycosidically linked (e.g., Gal 4 in the case of Gal β1→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 deduced from the position of H-1, H-5, and H-6 resonances For instance, the Lex and Lea

epitopes 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

H-5 signal of α-1,2-linked Fuc is observed at δ ~ 4.25–4.34 ppm These NMR data imply, 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 cannot

be 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 analyzed

by 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 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(β1-3)GlcNAc(β1-3)Gal(β1-4)GlcNAc or Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc, with additional Fuc or NeuAc attached to the terminal galactose units Since the presence of these peripheral monosaccharides affects essentially the chemical shifts of the terminal galactose, the anomeric protons can be assigned step-by-step

The compilation of 168 NMR spectra which are included in the review of Kamerling

et al (17) clearly shows that a spectrum is unique and can be used as an “identity

card.” This review presents the carbohydrate chains in a logical order and gives a classification according to the nature of the core, the nature of the backbone, and the peripherical monosaccharides Actually, the nature of the backbone can be often

di-Fig 2 Continued.

Analysis of Mucin-Type O-Linked Oligosaccharides 197

rectly deduced from the linkage of the fucose units, as discussed previously When the presence of these structural elements has been established, the number of possibilities decreases deeply, and a survey of the corresponding class of oligosaccharide-alditols

furnishes rapidly the structure of the compound Most of the O-glycans that constitute

the 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 glycan

sequences should be performed by 2D NMR spectroscopy

Fig 3 1 H-NMR spectra of oligosaccharide-alditols with the common tetrasaccharide N-1

(see Fig 2) variously substituted.

2.1.2.De novo Structural Elucidation of Glycan Sequences

by NMR Spectroscopy

Homonuclear correlated spectroscopy (COSY) provides information on directly coupled 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 be assigned Nevertheless, such an assignment is generally difficult when the cross peaks

Fig 3 Continued.

Fig 4 1 H-NMR spectrum of oligosaccharide-alditols with the common tetrasaccharide N-1

(see Fig 2) variously substituted.

Analysis of Mucin-Type O-Linked Oligosaccharides 199

occur around the diagonal Two-dimensional total correlation spectroscopy (2D 1 H-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 configuration

interrupt the assignment at the H-4 resonance Relayed COSY spectra have the advan-tage of successively assigning the 2 (COSY), 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-Gal

and one GlcNAc units For N-acetylglucosamine, H-2, H-3, and H-4 signals are

trip-Fig 4 Continued.

Klein 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., 1 III ) the protons 2 III , 3 III , and 4 III are

Analysis of Mucin-Type O-Linked Oligosaccharides 201

lets (transaxial hydrogens), whereas the same resonances of β-galactose are triplet, pseudo-doublet, and pseudo-singlet, respectively Fucose, which possesses an

α-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 the1H and 13C, 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 carbon In the example depicted in Fig 5, the crosspeak 4IIobserved at ~ 70 ppm corresponds to an unsubstituted carbon, in opposite to the signal 4III at 80 ppm The most elegant method for establishing the exact sequence of the oligosaccharide

is indisputably the heteronuclear multiple-bond correlation spectroscopy (HMBC) which relies the 1H and 13C via their 3JH,C coupling Unfortunately, the method is too

insensitive for being used systematically In the example given in Fig 5 these

ex-pected connectivities should be observed: 1IV→3IV, 5IV, 4III; 1III→3III, 5III, 3III; 1II→3II,

5II, 3I, and so on

2D Nuclear Overhauser effect spectroscopy (NOESY) or rotating-frame NOE spec-troscopy (ROESY) is generally used for establishing the sequence of carbohydrate chains Since the strongest NOE is not always between the protons connected to the linkage, the method may fail to establish the position of the glycosidic substitution

Figure 6 describes such an assignment A preliminary methylation analysis has shown

the presence of one terminal Gal, two O-3-substituted GalNAc, two O-4-substituted Gal, and one O-3-substituted GalNAc-ol The COSY experiment indicates that Gal

and GalNAc have β and α configuration, respectively The exact assignment of the five anomeric protons resulted from the connectivities 1IV/3V, 1V/4IV, 1IV/3III, 1III/4II, and 1II/3I

Fig 5 (continued) successively assigned The shape of the correlation peaks (triplet for H-2,

pseudo-doublet for H-3, pseudo-singlet for H-4) allows the estimatation of the coupling

con-stant: L (large) for J > 6 Hz; S (small) for J < 4 Hz For the monosaccharide unit III, we observe

J1,2> 6 Hz; J2,3> 6 Hz; J3,4<4 Hz; J4,5< 1 Hz These values demonstrate the following configu-ration of the ring protons: H-1, axial; H-2, axial; H-3, axial; H-4, equatorial, which are charac-teristic of a galactose residue in a β configuration With the same demonstration, units II, 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) For the β-Gal units, the 13 C-atom resonances 2 II , 3 II ,

2 III , and 4 III are deshielded from 3 to 10 ppm, as compared to the standard values observed for β-methylgalactoside On the contrary, the signal 3 III and 4 II possess normal values (nonsubsti-tuted hydroxyl group at position 3 and 4, respectively).

Klein et al.

Fig 6 COSY (left) and ROESY (right) NMR spectra of a hexasaccharide-alditol isolated from the oviducal mucin of Rana palustris 2II , proton H-2 of monosaccharide unit II For the COSY spectrum, the carbohydrate units were identified on the basis of the set of the vicinal

coupling constants as described in Fig 5 For the ROESY spectrum, the correlations 1VI → 3 V , 1 V → 4 IV , 1 IV → 3 III, 1 III → 4 II

, and 1 II → 3 I

clearly indicate the sequence of the oligosaccharide and confirm the assignment of each anomeric proton.