Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion ofthe cDNA encoding GM-CSF with the cDNA encoding EPO followed bytransfection of the hybrid gene into CHO cells. The oligonucleotideconstruct incorporated a spacing sequence between the two individual cDNAswhich encodes eight amino acids constituting a linker peptide intended toseparate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein wassubmitted to a detailed structural characterization including theverification of the entire amino acid sequence, the assignment of thedisulfide bridges pattern, the identification of the glycosylation sitesand the definition of the glycosidic moiety, including site-specificity.Partial processing of the C-terminal Arg residue and the occurrence ofN-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established.Moreover, O-glycosylation at Ser257 and at the N-terminal region was alsodetected. A large heterogeneity was observed in the N-glycans due to thepresence of differently sialylated and fucosylated branched complex typeoligosaccharides whereas O-linked glycans were constituted by GalGalNAcchains with a different number of sialic acids. The disulfide bridgespattern was established by direct FABMS analysis of the proteolytic digestsor by ESMS analysis of HPLC purified fractions. Pairing of the eightcysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, andCys160-Cys164. This S-S bridges pattern is identical to that occurring inthe individual natural GM-CSF and EPO, thus showing that the two proteinmoieties in MEN 11300 can independently acquire their nativethree-dimensional structure.