Protein Protocols Protein Protocols

Lectin-Binding Assay for Glycoanalysis 795
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A Lectin-Binding Assay for the Rapid Characterization
of the Glycosylation of Purified Glycoproteins
Mohammad T. Goodarzi, Angeliki Fotinopoulou, and Graham A. Turner
Introduction
Most proteins have carbohydrate chains (glycosylation) attached covalently to various
sites on their polypeptide backbone. These posttranslational modifications, which
are carried out by cytoplasmic enzymes, confer subtle changes in the structure and
behavior of a molecule, and their composition is very sensitive to many environmental
influences (1–3). There is increasing interest in determining the glycosylation of a
molecule because of the importance of glycosylation in affecting its reactivity (1,4,5).
This is particularly true in the production of therapeutic glycoproteins by recombinant
methods, in which glycosylation can be determined by the type of host cell used or the
production process employed (2). Glycosylation is also important in disease situations
in which changes in the carbohydrate structure can be involved in the pathological
processes (3,6,7,8). Unfortunately, the glycosylation of proteins is very complex; there
are variations in the site of glycosylation, the type of amino acid–carbohydrate bond,
the composition of the chains, and the particular carbohydrate sequences and linkages
in each chain (3,4). In addition, within any population of molecules there is considerable
heterogeneity in the carbohydrate structures (glycoforms) that are synthesized at
any one time (1). This is typified by some molecules showing increased branching,
reduced chain length, and further addition of single carbohydrate moieties to the internal
chain.
To unravel completely the complexities of the glycosylation of a molecule is a substantial
task, which requires considerable effort and resources. However, in many situations
this is unnecessary, because only a limited amount of information, on a single or
a group of structural features, is needed. Lectins can be useful for this purpose. These
substances are carbohydrate binding proteins with particular specificity (9). Although
their specificity is not absolute, there is usually one carbohydrate or group of carbohydrates
to which the lectin binds with a higher affinity than the rest of the group. The major
carbohydrate specificity of a number of commonly used lectins is shown in Table 1.
Lectins have previously been used for investigating glycoprotein glycosylation by
incorporating them into existing technologies such as affinity chromatography, blotting,
and electrophoresis. Although these modifications give workable methods, they
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
795