Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Abstract

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.

Introduction

The properties of a protein are largely determined by its amino acid sequence. However, protein characteristics are also modified and regulated by a large number of post-translational modifications, taking place during or after synthesis of the polypeptide chain (Walsh et al., 2005). One of the most common post-translational modifications is glycosylation and approximately half of all human proteins are estimated to be glycoproteins (Wong, 2005). Protein glycosylation is a conserved mechanism that occurs in yeast, plants and animals (Lommel and Strahl, 2009). Simple forms of glycosylation have also been identified in bacteria (Nothaft and Szymanski, 2010). Glycosylation are enzymatic processes where glycans are added to specific amino acids in the polypeptide chain. Two types of glycosylation, N-linked and O-linked, occur in proteins. N-linked glycosylation starts as a co-translational process in the endoplasmic reticulum (ER), where a branched presynthesized oligomeric glycan structure is attached to the nitrogen of an aspargine in the protein chain (Yan and Lennarz, 2005). The glycan structure is then trimmed before the protein is transferred to the Golgi apparatus (GA) where the glycan structure is further modified. O-linked glycosylation is a post-translational process occurring in the GA (Peter-Katalinic, 2005). Single monosaccharides are attached to the hydroxyl group of serine or threonine residues. The glycan structures are subsequently built up by addition of individual monosaccharides. In addition to the two major glycosylation pathways, glycans can also be attached to arginine, tyrosine, hydroxylysine, hydroxyproline and tryptophan residues (Spiro, 2002). These modifications are less common and restricted to specific proteins. Protein glycosylation can be very heterogeneous, resulting in a large number of isoforms of the protein (Hua et al., 2011). Potential glycosylation sites can be either occupied or unmodified, and each site can be occupied by a different glycan structure in different protein molecules.

Although, glycosylation was long considered as an unimportant protein decoration, it is now clear that it has important functions. Glycosylation affects protein folding, stability, solubility, protein–protein interactions and in vivo bioavailability, biodistribution, pharmacokinetics and immunogenecity (Kaushik et al., 2011, Kayser et al., 2011, Li and d’Anjou, 2009, Oberg et al., 2011, Opanasopit et al., 2001). In general the activity of a protein is determined by its primary amino acid sequence however, there are several examples where glycosylation affects and regulates the activity (Rajagopalan et al., 2010, Straumann et al., 2006, Su et al., 2010). Impaired or changed protein glycosylation is associated with a number of human pathologies, including, rheumatoid arthritis, Leroy disease, and leukocyte-adhesion deficiency type II (Gornik and Lauc, 2008, Koscielak, 1995). Alterations in protein glycosylation are frequently associated with different cancers, although the function or implication of those changes mostly remains unclear (Dennis et al., 1999, Orntoft and Vestergaard, 1999).

Most proteins used for in vitro and in vivo studies today are produced as recombinant proteins in various cell culture based expression systems. Widely used expression systems are, in addition to bacteria, where little post-translational modifications occur, yeast, insect cells and different mammalian cell lines. It is well known that the glycosylation structures are different between mammalian, yeast and insect cells (Brooks, 2006). However, it is much less known what differences exist between mammalian cell lines. Two frequently used mammalian cell lines are HEK and CHO. HEK is a cell line originally derived from Human Embryonic Kidney tissue. HEK cells are easy to grow and transfect, and transient transfection frequently gives good expression levels, which has made this cell line widely used in research. CHO is derived from Chinese Hamster Ovary. This cell line is frequently used for expression after stable transfections, it has a good long-term stable gene expression with high expression levels. For transient expression CHO is more difficult to transfect and the expression levels are frequently lower compared to transiently transfected HEK cells.

For this study 12 proteins containing different numbers of potential N- and O-linked glycosylation sites were selected and each one was expressed and purified from HEK and CHO cells. The purified proteins were analyzed by SDS-PAGE, gel IEF and/or capillary IEF. To verify the contribution of glycosylation to the differences in protein patterns detected the proteins were treated with deglycosylating enzymes and reanalyzed after the treatment. For the more in depth analyses subsets of the proteins were analyzed by mass spectrometry and released N-linked glycans were analyzed by capillary gel electrophoresis.