Glycosylation in rhgm-csf and its effect on pharmacological properties

Roghayeh Shirvani,1,* Mohammad barshan tashnizi,2 Maryam shahali,3

1. Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran
2. Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran
3. Production and Research Complex, Pasteur Institute of Iran

Abstract

Introduction

Glycosylation is one of the most common post-translational modifications occurring in more than 50% of eukaryotic proteins. glycosylation affects the properties of pharmaceutical proteins, including pharmacokinetics, in-vivo half-life, immunogenicity and toxicity. granulocyte macrophage-colony stimulating factor (hgm-csf) or csf2 is a type of cytokine produced by recombinant dna technology. this glycoprotein is naturally secreted as a growth factor from the immune cells by stimulating the bone marrow stem cells. gm-csf causes the differentiation of white and red blood cells and platelets. studies conducted so far have revealed the biological and therapeutic importance of this protein for the treatment of cancer and immunodeficiency diseases. the human gm-csf protein has 127 amino acids and the final weight is 23kda. the protein has o-linked glycosylation in the amino acid residues of 22,24,26,27, also has two n-linked glycosylation at positions of 44 and 45. in recombinant production, depending on the expression system and the bioprocess aspects, glycosylated forms of varying molecular weight can range from 14 to 60 kda. the lowest molecular weight is e. coli derived form that is non-glycosylated. the 16 isoforms of this protein have been observed by cho expression while the molecular weight is between 14.5 and 35 kda, due to differences in sugar content. the recombinant yeast derived forms are glycosylated and sometimes hyperglycosylated so have higher molecular weight than natural gm-csf.

Methods

Data obtained from research articles by search on sciencedirect, pubmed, scopus and google scholar search engines. the results of the papers from 1978-2017 were reviewed with a comparative approach on the effect of glycosylation on the pharmacological properties of the recombinant human gm-csf.

Results

In the early years studies by donahue et al in 1986, it has been proven that different levels of glycosylated protein exhibit different kinetics of plasma distribution. for example, gm-csf with high molecular weight secreted in far from infected area is more effective than smaller forms because it has more stability in the pathway of the bloodstream to reach the bone marrow and ultimately leads to more neutrophils production. elimination of n-linked oligosaccharides from yeast and cho derived recombinant gm-csf with deglycosylation enzyme treatment, increased the immunoactivity about, 4 and 8 folds, respectively. it was also found that the glycosylated form has 6 times less effect on immune cell proliferation. the protein expressed by pichia pastoris expression system was seen from 28-35 kda. hyperglycosylated forms of 38-76 kda were observed in the expression of this protein with yarrowia lipolytica. the biological activity of 17 kda was 9 times higher than the hyperglycosylated forms.

Conclusion

Increasing immune activity after removing glycosylation suggests that carbohydrates seem to prevent protein interactions with specific immunoglobulins. also, receptor binding studies showed that the receptor had a lower affinity for hyperglycosylated molecules. it seems that the sugar units would interfere with the interaction of this protein with its receptor. this may be the cause of bioactivity reduction in glycosylated forms. despite the negative effects of glycosylation on activity, based on protein stability studies, it was assumed that the glycosylation level of this protein may be involved in increasing the half-life in the serum. plasma pharmacokinetic studies have shown that different forms of this protein are distributed differently in the body, so the stability of this protein affects the drugs reach to the target cells population. nowadays, sargramostim (leukine) as yeast derived recombinant hgm-csf is the only commercially available form of this pharmaceutical protein. therefore the glycoproteins with a molecular weight 15.5-20 kda are preferred to the non-glycosylated e.coli derived rhgm-csf. it can be concluded that due to the pharmacokinetic advantages of this glycoprotein, such as protection against proteolysis, increased serum half-life, improved stability and solubility, its biological activity reduction compared with non-glycosylated form, have been ignored.

Keywords

rhgm-csf, glycosylation, glycoproteins, cytokine, bioactivity assay