Protein Protocols Protein Protocols

834 Weitzhandler et al.
alditols (10,11), gives an isocratic baseline separation of GlcNAc, GalNAc, fucose,
mannose, glucose, and galactose, and simultaneously resolves many neutral oligosaccharides.
This separation extends the usefulness of the CarboPac MA1 column to the
assay of reducing monosaccharides released by exoglycosidases. In the following, an
assay of exposed GlcNAc after β-N-acetylhexosaminidase treatment of a variety of
glycoconjugates is shown.
To determine the suitability of HPAEC-PAD and the MA1 column for analyzing
both released monosaccharide and oligosaccharide products in a single analysis, we
subjected an asialo agalacto biantennary oligosaccharide standard (Table 1, structure
3; Fig. 1A, peak 3) and an asialo agalacto tetraantennary oligosaccharide standard
(Table 1, structure 5; Fig. 1B, peak 5) to Jack bean β-N-acetylhexosaminidase digestion.
In addition to differences in numbers of antennae (2 vs 4) and retention times
(22.3 vs 25.2 min), these two oligosaccharides differ in that the tetraantennary oligosaccharide
has terminal GlcNAc linked to mannose β(1→4) and β(1→6) in addition
to the β(1→2) linkages present in the biantennary oligosaccharide standard.
The complete disappearance of the asialo agalacto biantennary substrate is indicated
by the disappearance of peak 3 (Fig. 1A; compare dashed vs solid line in bottom
tracing). The complete disappearance of the asialo agalacto tetraantennary substrate is
indicated by the disappearance of peak 5 (Fig. 1B; compare dashed vs solid line in
bottom tracing). The expected digestion products of both structures 3 and 5 would be
the released monosaccharide GlcNAc, (see Fig. 1A,B; peak at 15.6 min) and the
released, shortened oligosaccharide product, Man 3GlcNAc 2 (Table 1, structure 1;
see Fig. 1A,B, peak at 17.5 min).
In addition to being useful for monitoring the β-N-acetylhexosaminidase release of
terminal GlcNAc from isolated oligosaccharides, HPAEC-PAD and the MA1 column
can be used to directly monitor the presence of terminal GlcNAc on glycoproteins.
Such an assay could be useful for monitoring terminal carbohydrate modifications in
therapeutic glycoproteins; these modifications have been shown to affect the stability
and efficacy of therapeutic glycoproteins (12). The β-N-acetylhexosaminidase release
of terminal GlcNAc from a monoclonal IgG is shown in Fig. 2A (bottom tracing; compare
solid vs dashed line). Additionally, the absence of contaminating exoglycosidases
is apparent by the absence of release of any other monosaccharides.
To assess whether the released GlcNAc was derived from a GlcNAc-terminated
N-linked oligosaccharide, the monoclonal IgG was treated with peptide-N-glycosidase
(PNGase F), an amidase that nonspecifically releases N-linked oligosaccharides from
glycoproteins. The N-linked oligosaccharide map of the monoclonal IgG is shown in
Fig. 2B (bottom tracing; solid line). The major PNGase F product peak eluted between
18.5 and 19 min with a retention time similar to a core fucosylated asialo agalacto
biantennary oligosaccharide standard (18.7 min, see Table 1, structure 4; see also
Fig. 2B, peak 4). The second PNGase F released peak had a retention time of 21 min
and could represent a monogalactosylated, biantennary oligosaccharide with core
fucosylation, as has been reported in other monoclonal IgGs (7). To assess whether the
PNGase F released oligosaccharides were terminated with GlcNAc, the PNGase F
released oligosaccharides were treated with β-N-acetylhexosaminidase.