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Abstract
A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each
protein molecule may be associated with an array of oligosaccharide structures. The
overall range of glycoforms can have a variety of different biophysical and biochemical
properties, although details of structure-function relationships are poorly understood,
because of the microheterogeneity of biological samples. Hence, there is clearly a
need for synthetic methods that give access to natural and unnatural homogeneously
glycosylated proteins. The synthesis of novel glycoproteins through the selective
reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed
by site-directed mutagenesis at desired glycosylation sites has been developed. This
provides a general method for the synthesis of homogeneously glycosylated proteins
that carry saccharide side chains at natural or unnatural glycosylation sites. Here,
we have shown that the approach can be applied to the glycoprotein hormone erythropoietin,
an important therapeutic glycoprotein with three sites of N-glycosylation that are
essential for in vivo biological activity.
Wild-type recombinant erythropoietin and three mutants in which glycosylation site
asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys,
His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields
of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides
could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins,
the potential side reaction of the other four cysteine residues (all involved in disulfide
bonds) were not observed. Yield of glycosylation was generally about 50% and purification
of glycosylated protein from non-glycosylated protein was readily carried out using
lectin affinity chromatography. Dynamic light scattering analysis of the purified
glycoproteins suggested that the glycoforms produced were monomeric and folded identically
to the wild-type protein.
Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural
glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even
in the presence of two disulfide bonds. The findings provide the basis for further
elaboration of the glycan structures and development of this general methodology for
the synthesis of semi-synthetic glycoproteins.