Protein Protocols Protein Protocols

Sequencing Heparan Sulfate Saccharides 893
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Sequencing Heparan Sulfate Saccharides
Jeremy E. Turnbull
1. Introduction
The functions of the heparan sulfates (HSs) are determined by specific saccharide
motifs within HS chains. These sequences confer selective protein binding properties
and the ability to modulate protein activities (1,2). HS chains consist of an alternating
disaccharide repeat of glucosamine (GlcN; N-acetylated or N-sulfated) and uronic acid
(glucuronic [GlcA] or iduronic acid [IdoA]). The initial biosynthetic product containing
N-acetylglucosamine (GlcNAc) and GlcA is modified by N-sulfation of the GlcN,
ester (O)-sulfation (at positions 3 and 6 on the GlcN and position 2 on the uronic acids)
and by epimerization of GlcA to IdoA. The extent of these modifications is incomplete
and their degree and distribution varies in HS between different cell types. In HS chains
N- and O-sulfated sugars are predominantly clustered in sequences of up to eight disaccharide
units separated by N-acetyl-rich regions with relatively low sulfate content (3).
Sequence analysis of HS saccharides is a difficult analytical problem and until
recently sequence information had been obtained for only relatively short saccharides
from HS and heparin. Gel chromatography and high-performance liquid chromatography
(HPLC) methods have been used to obtain information on disaccharide composition
(3,4). Other methods such as nuclear magnetic resonance (NMR) spectroscopy
and mass spectroscopy (5–9) have provided direct sequence information, but are difficult
for even moderately sized oligosaccharides and in the case of NMR requires large
amounts of material (micromoles). This situation has changed rapidly in the last few
years with the availability of recombinant exolytic lysosomal enzymes. These
exoglycosidases and exosulfatases remove specific sulfate groups or monosaccharide
residues from the nonreducing end (NRE) of saccharides (10). They can be employed
in combination with polyacrylamide gel electrophoresis (PAGE) separations to derive
direct information (based on band shifts) on the structures present at the nonreducing
end of GAG saccharides (11; see Fig. 1 for an example).
Integral glycan sequencing (IGS), a PAGE-based method using the exoenzymes,
was recently developed as the first strategy for rapid and direct sequencing of HS and
heparin saccharides (11). Its introduction has been quickly followed by a variety of
similar approaches using other separation methods including HPLC and matrix-assisted
laser desorption (MALDI) mass spectrometry (12–14). An outline of the IGS sequenc-
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
893