Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosyl ated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a vari ety of different biophysical and biochemical properties, although details o f structure-function relationships are poorly understood, because of the mi croheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously g lycosylated proteins. The synthesis of novel glycoproteins through the sele ctive reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sit es has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at na tural or unnatural glycosylation sites. Here, we have shown that the approa ch can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essen tial for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which gl ycosylation site asparagine residues had been changed to cysteines (His(10) -WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were ove rexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Che mical 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 tall involved i n disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated pr otein 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. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn drop Cys mutations at natural glycosylation sites can be glycosylated using beta -N-glycosyl iodoacetamides even in the presence of two disulfide bonds. Th e findings provide the basis for further elaboration of the glycan structur es and development of this general methodology for the synthesis of semi-sy nthetic glycoproteins. (C) 2001 Elsevier Science Ltd. All rights reserved.