324
P. M. RUDD et al.
ence to a dextran ladder, are used to predict structure based on experimentally
determined incremental values for the addition of all monosaccharide residues to
basic core oligo saccharides. Thus preliminary structural assignments may be
made from the profile of a single HPLC run (Fig. 23.2).
The preliminary assignments are confirmed by exoglycosidase digestions.
Arrays of highly specific enzymes are used to digest aliquots of the whole pool of
sugars simultaneously. The products of such digestions are identified using the
same HPLC system, based on the incremental value for each type of monosaccharide residue and the specificity of the enzymes in the arrays. Typically, aliquots of
the entire glycan pool are sequenced simultaneously using a standard panel of
five enzyme arrays, after which the products are analysed using the same HPLC
system (Fig. 23.2). Any less common or novel structures, such as sulphated sugars, are highlighted since these will not be digested to a common tri-mannosyl
N-linked glycan core by any of the five standard enzyme arrays.
4
Glycoproteins exist as Populations of Glycoforms
and the 3D Structure of the Individual Protein
is a Factor which Influences its own Glycosylation Pattern
Glycoproteins generally exist as populations of glycosylated variants (glycoforms)
of a single polypeptide (Review: Rudd and Dwek 1997). Although the same glycosylation machinery is available to all proteins which enter the secretory pathway
in a given cell, most glycoproteins emerge with a characteristic glycosylation pattern and heterogeneous populations of glycans at each glycosylation site. The
local protein structure plays a key role in glycan processing (Rudd et aI., in
press). In the immunoglobulin family for example, N-linked glycans are acquired
by the transfer of the oligosaccharide precursor, Glc3Man9GlcNAc2, to some Asn
residues in the Hand L chains. This event takes place co-translationally in the
endoplasmic reticulum (ER) before the protein is fully folded. The glucose residues and some mannose residues are subsequently trimmed in the ER, where
immunoglobulin monomers are assembled from Hand L chains (Bole et aI.,
1986). In some cases, such as IgA and IgM, monomers assemble further in the ER
forming dimers and pentamers, respectively.
The assembled immunoglobulins are transferred to the Golgi apparatus where
the N-linked oligomannose sugars are processed to complex glycans and 0linked glycans are attached to some accessible Ser or Thr residues, beginning
with the addition of GaINAc. The processing of N-linked sugars and the attachment and processing of O-glycans therefore depends both on the local 3D structure of the immunoglobulin fold around the glycosylation sites and on the structure of the fully assembled multimers.
P. M. RUDD et al.
ence to a dextran ladder, are used to predict structure based on experimentally
determined incremental values for the addition of all monosaccharide residues to
basic core oligo saccharides. Thus preliminary structural assignments may be
made from the profile of a single HPLC run (Fig. 23.2).
The preliminary assignments are confirmed by exoglycosidase digestions.
Arrays of highly specific enzymes are used to digest aliquots of the whole pool of
sugars simultaneously. The products of such digestions are identified using the
same HPLC system, based on the incremental value for each type of monosaccharide residue and the specificity of the enzymes in the arrays. Typically, aliquots of
the entire glycan pool are sequenced simultaneously using a standard panel of
five enzyme arrays, after which the products are analysed using the same HPLC
system (Fig. 23.2). Any less common or novel structures, such as sulphated sugars, are highlighted since these will not be digested to a common tri-mannosyl
N-linked glycan core by any of the five standard enzyme arrays.
4
Glycoproteins exist as Populations of Glycoforms
and the 3D Structure of the Individual Protein
is a Factor which Influences its own Glycosylation Pattern
Glycoproteins generally exist as populations of glycosylated variants (glycoforms)
of a single polypeptide (Review: Rudd and Dwek 1997). Although the same glycosylation machinery is available to all proteins which enter the secretory pathway
in a given cell, most glycoproteins emerge with a characteristic glycosylation pattern and heterogeneous populations of glycans at each glycosylation site. The
local protein structure plays a key role in glycan processing (Rudd et aI., in
press). In the immunoglobulin family for example, N-linked glycans are acquired
by the transfer of the oligosaccharide precursor, Glc3Man9GlcNAc2, to some Asn
residues in the Hand L chains. This event takes place co-translationally in the
endoplasmic reticulum (ER) before the protein is fully folded. The glucose residues and some mannose residues are subsequently trimmed in the ER, where
immunoglobulin monomers are assembled from Hand L chains (Bole et aI.,
1986). In some cases, such as IgA and IgM, monomers assemble further in the ER
forming dimers and pentamers, respectively.
The assembled immunoglobulins are transferred to the Golgi apparatus where
the N-linked oligomannose sugars are processed to complex glycans and 0linked glycans are attached to some accessible Ser or Thr residues, beginning
with the addition of GaINAc. The processing of N-linked sugars and the attachment and processing of O-glycans therefore depends both on the local 3D structure of the immunoglobulin fold around the glycosylation sites and on the structure of the fully assembled multimers.