Tài liệu Báo cáo khóa học: Determination by electrospray mass spectrometry and 1H-NMR spectroscopy

Determination by electrospray mass spectrometry and 1

H-NMR

spectroscopy of primary structures of variously fucosylated neutral

oligosaccharides based on the iso-lacto-N-octaose core

Heide Kogelberg1

, Vladimir E. Piskarev2

, Yibing Zhang1

, Alexander M. Lawson1 and Wengang Chai1

1

MRC Glycosciences Laboratory, Imperial College Faculty of Medicine, Northwick Park Institute for Medical Research, Harrow,

Middlesex, UK; 2

Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia

We have isolated a nonfucosylated and three variously

fucosylated neutral oligosaccharides from human milk that

are based on the iso-lacto-N-octaose core. Their structures

were characterized by the combined use of electrospray

mass spectrometry (ES-MS) and NMR spectroscopy. The

branching pattern and blood group-related Lewis deter￾minants, together with partial sequences and linkages of

these oligosaccharides, were initially elucidated by high￾sensitivity ES-MS/MS analysis, and then their full structure

assignment was completed by methylation analysis and

1

H-NMR. Three new structures were identified. The

nonfucosylated iso-lacto-N-octaose, Galb1–3GlcNAcb1–

3Galb1–4GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc,

has not previously been reported as an individual oligo￾saccharide. The monofucosylated and trifucosylated

iso-lacto-N-octaose,Galb1–3GlcNAcb1–3Galb1–4(Fuca1–3)

GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc and Galb1–

3(Fuca1–4)GlcNAcb1–3Galb1–4(Fuca1–3)GlcNAcb1–

6[Galb1–3(Fuca1–4)GlcNAcb1–3]Galb1–4Glc, both con￾taining an internal Lex epitope, are also novel structures.

Keywords: electrospray mass spectrometry; human milk;

oligosaccharide; NMR.

A role for carbohydrates in cellular events has long been

hypothesized, although strong evidence for this has only

emerged over the last two decades. Awareness of the

biological function of oligosaccharide chains in glycopro￾teins, glycolipids and proteoglycans has intensified as an

increasing number of examples have been reported that

reveal that carbohydrate structures participate in various

biological events in addition to modifying protein function.

One of the early demonstrations of the role of carbo￾hydrates in recognition was binding of the influenza virus to

red blood cells via sialic acid [1], and later by work on the

chemical basis of the antigenicity of polysaccharides and

of the well-known ABO (H) blood-group system [2,3],

in which specificity is determined by oligosaccharide

sequences. Carbohydrates are well placed to act in cellular

recognition as many cells are surrounded by an oligosac￾charide layer made from cell-associated glycoconjugates,

which often overshadows protein and lipid components on

the cell surface. Specific oligosaccharide sequences, such

as the type 1 (Galb1–3GlcNAc)/type 2 (Galb1–4GlcNAc)

chains and the blood group-related antigens bearing the

H (Fuca1–2Galb1–3/4GlcNAc), Lewisa [Lea

, Galb1–

3(Fuca1–4)GlcNAc] and Lewisx [Lex

, Galb1–4(Fuca1–

3)GlcNAc] determinants, occur naturally as structural

elements of free oligosaccharides or on the carbohydrate

chains of glycoproteins and glycolipids and comprise

recognition motifs for cell–cell and cell–matrix interactions

[4,5].

Human milk is a unique source of diverse oligosaccha￾rides, and more than 80 have been isolated and sequences

assigned [6]. Many of these structures are closely related to

the carbohydrate chains of glycoproteins and glycolipids [7].

These diverse oligosaccharide sequences may also serve as

cell differentiation and tumour antigens [5]. Milk oligosac￾charides are considered to play a part in the inhibition of

bacterial adhesion to epithelial surfaces, as they are able to

mimic the binding epitope of the epithelial receptor [8].

Also, milk contains oligosaccharides that resemble structures

recognized by the cell–cell adhesion molecules, the selectins,

suggesting a role in inflammatory processes [8,9]. Human

milk has also been used as a rich source of oligosaccharides

to map the fine binding specificity of E-selectin [10].

In contrast with oligonucleotides and peptides, oligosac￾charides can be branched, and hence a relatively simple set

of monosaccharides can form a huge number of complex

structures. A greater degree of structural complexity

produced by branching is the norm for naturally occurring

carbohydrates, and often a branched sequence carrying two

or more recognition motifs is more potent [11,12]. Free

oligosaccharides from human (milk, urine and infant faeces)

have a common lactose (Galb1–4Glc) core. It can be

extended, for example, at the 4-position of the Gal as a

linear sequence or at its 3,6-positions as a branched

sequence. The linear and branched chains are often

Correspondence to W. Chai, MRC Glycosciences Laboratory,

Imperial College Faculty of Medicine, Northwick Park Institute for

Medical Research, Watford Road, Harrow, Middlesex HA1 3UJ,

UK. Fax: + 44 20 8869 3253, Tel.: + 44 20 8869 3252,

E-mail: [email protected]

Abbreviations: CID, collision-induced dissociation; ES-MS, electro￾spray mass spectrometry; iLNO, iso-lacto-N-octaose; Lea

, Lewis a;

Lex

, Lewis x; PMAA, partially methylated alditol acetate; rOe,

rotating frame nuclear Overhauser enhancement.

(Received 4 December 2003, accepted 3 February 2004)

Eur. J. Biochem. 271, 1172–1186 (2004)
FEBS2004 doi:10.1111/j.1432-1033.2004.04021.x