A lot of recombinant glycoproteins, and erythropoietin, and tissue plasminogen activator (tPA) are among them, are used as therapeutics aimed at mimicking the native human protein. The efficient and safe use of glycoprotein drugs requires determining the mixed structures of their carbohydrate moieties because the attachment of oligosaccharides (glycans) to glycoproteins has proven to influence various properties of glycoproteins.
The structures of oligosaccharides vary in different expressions especially from those in the native human structures. Lactoferrin is an iron-binding glycoprotein that is expressed in most human bodily fluids, including saliva, bile, and most highly in milk. The goal of this experiment is to compare the glycosylation of the native lactoferrin isolated from human milk and the same protein as recombinantly expressed in rice.
The method of the experiment is the analysis of the N-linked oligosaccharides after releasing them from the glycoproteins enzymatically. After the reduction and alkylation of the glycoproteins, they are digested with trypsin. N-Glycosidase F (PNGase F) and N-glycosidase A (PNGase A) will be used as enzymes for releasing oligosaccharides from the glycopeptides. The technique of the capillary carbon liquid chromatography (LC) combined with an Electrospray Ionisation (ESI) mass spectrometer is used for the analysis of the isolated glycans.
The post-translational modifications (PTMs), particularly glycosylation can affect the structure, stability and functions of the glycoproteins. Dealing with glycosylation requires considering the main characteristics of the process and possible results of the experiment for enhancing its effectiveness. Sawa et al (2006) noted that “glycosylation is an important posttransnational modification affecting >50% of eurokaryotic proteins” (p. 12371).
Like many other eurokariotic proteins, human lactoferrin is modified by N-linked glycosylation which consists of the three possible sites, namely Asn 138, Asn 479 and Asn 624. The study of Berkel et al (1996) has determined the extent to which each of the three sequons is used, concluding that “glycosylation at Asn479 in the Asn 138 mutant as well as in natural hLF decreases the glycosylation efficiency at Asn624” (p. 121). Along with the glycosylation heterogeneity, this relationship needs to be taken into consideration for making the analysis of the results more comprehensive.
The release of oligosaccharides from glycopeptides presupposes elucidation of the structure of the glycan component. The sequential isolation of individual monosaccharides is required for completing the release. The PNGase F is recognized as the most effective method of removing the oligosaccharides from the glycoproteins. However, the oligosaccharides commonly found in the glycoproteins of plants and parasitic worms can be resistant to this method.
In these cases PNGase A can be used for removing the oligosaccharides which are resistant to the PNGase F. The study by Moloney (2000) has demonstrated that particular types of the oligosaccharides released from the human cells can also be resistant to the PNGase F. Considering the fact that the native lactoferrin is compared to the same protein as recombinantly expressed in rice, both methods need to be used in the experiment. A comprehensive analysis of the achieved results requires implementing the recent advances in mass spectrometry such as capillary carbon liquid chromatography (LC) (Pan et al 2010).
The glycoproteins have become the important research targets related to the spheres of disease diagnosis, prognosis and reaction of the human organism to the therapeutic drugs containing the recombinant glycoproteins. The comparison of native lactoferrin and the same protein as recombinantly expressed in rice can be helpful for safer and more effective use of the glycoprotein drugs.
References
Berkel, H., Veen, H., Ceerts, M., Boer, H. and Nuijens, J. (1996). Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin : a study with glycosylation-site mutants. Biochem J., 319: p. 117 – 122.
Moloney, D., Shair, L., Lu, F., Xia, J., Locke, R. (2000). Mammalian notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor0like modules. The Journal of Biological Chemistry, 275: 9604-9611.
Pan, S., Chen, R., Aebersold, R. and Brentnall, T. (2010). Mass spectrometry based glycoproteomics – From a proteomics perspective. Molecular and Cellular Proteomics. 10.
Sawa, M., Hsu, T., Itoh, T., Sugiyama, M., Hanson, S., Vogt, P. (2006). Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. PNAS, 103 (33): p. 12371 – 12376.