N‐ and O‐linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte‐macrophage colony‐stimulating factor secreted by a Chinese hamster ovary cell line

Abstract

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Dm 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri-and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal a1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found a2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N-and C-termini integrity of the GM-CSF preparations.

Synthesis of granulocyte–macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

“…Interestingly, neither the position nor the extent of O-linked glycosylation has been unambiguously established. Studies suggest a range from two (Ser 7 and Ser 9 /Thr 10 ) to four (Ser 5 , Ser 7 , Ser 9 , and Thr 10 ) sites of glycosylation (17). The tertiary structure is strongly influenced by two cross-linked disulfide bonds.…”

The Glycosylation and in Vivo Stability of Human Granulocyte-Macrophage Colony-Stimulating Factor Produced in Rice Cells

“…2). 13,14) After TFMS treatment, its molecular weight was shifted to 15 kDa, 14) showing that 9-13 kDa of glycans had been removed (Fig. 2).…”

“…DISCUSSION hGM-CSF has two potential N-glycosylation sites (Asn27, Asn37) and three potential O-glycosylation sites (Ser7, Ser9, Thr10). 4,12,13) It is known that degrees of glycosylation differ according to cell type and that in vivo stability increases with N-glycan content. 14) Terminal sialic acids are important for the in vivo stability of glycoproteins.…”

“…14) N-Glycosylation of hGM-CSF has an important role in prolonging its half-life in the blood stream. 13,14) Therefore, the in vivo stability of heavily glycosylated animal cell-derived rhGM-CSF is superior to that of the yeast and Escherichia coli-derived forms. 14) Transgenic plant-derived rhGM-CSF has been produced in tobacco cells, 8) tomato cells, 10) sugarcane 11) and rice cells.…”

“…[8][9][10][11] hGM-CSF has two potential N-glycosylation sites (Asn 27, Asn 37) and three potential O-glycosylation sites (Ser 7, Ser 9, Thr 10). 4,12,13) Escherichia coli-derived rhGM-CSF has a molecular weight of 14.5 kDa and is nonglycosylated. 7) Yeast-derived rhGM-CSF has a molecular weight of 16-18 kDa and is O-glycosylated, which contains leucine instead of proline at position 23 of mature form of wild-type hGM-CSF.…”