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 of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.
Amoresano
A
A
Centro Internazionale di Servizi di Spettrometria di Massa, Via Pansini 5, 80131 Napoli, Italy, Istituto di Scienza dell'Alimentazione del CNR, Avellino, Italy, Centro di Biotecnologie Menarini Ricerche S.p.A., Pomezia, Italy.
Andolfo
A
A
Siciliano
R A
RA
Mele
A
A
Coscarella
A
A
De Santis
R
R
Mauro
S
S
Pucci
P
P
Marino
G
G
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D004220:Disulfides / Q000737:chemistry; D004921:Erythropoietin / Q000737:chemistry*; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D004220:Disulfides; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor