Individual Glycoforms Research Articles

Effects of glycosylation on protein structure and dynamics in ribonuclease B and some of its individual glycoforms.

In an attempt to elucidate the role of carbohydrates on protein structure and dynamics in glycoproteins, ribonuclease B (RNase B), containing a single glycosylation site at Asn34, has been investigated and compared with the enzyme in the unglycosylated form (RNase A). RNase B consists of five glycoforms: Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2 (Man5-9GlcNAc2). The individual glycoforms Man1GlcNAc2 (synthetic) and Man5GlcNAc2 of RNase B have been studied to ascertain any specific effects of the different oligosaccharides. NMR measurement of amide-proton/deuterium exchange rates shows that glycosylation of the enzyme leads to the protection of amide-proton resonances from solvent exchange for a large number of residues, both in the vicinity of the glycosylation site (e.g. residues 29-34 and 35) and remote from it (e.g. residues 57-61 and 75-76). In addition, residues 10-13, 32, 34 and 35, which are observed to be protected from exchange as a result of glycosylation in the individual glycoforms Man1Glc-NAc2-RNase and Man5GlcNAc2-RNase (when compared with RNase A) are less protected in RNase B. This additional protection in the glycoforms Man1GlcNAc2-RNase and Man5GlcNAc2-RNase may arise from steric hindrance between the oligosaccharide and protein reducing solvent accessibility. The rates of solvent exchange of amide protons for residues 10-13, 32, 34 and 35 are dependent on the oligosaccharide moiety. The average amide-proton/deuterium exchange rate in Man6-9Glc-NAc2-ribonucleases for residues 10-13 and 35 is approximately three times greater than Man5Glc-NAc2-ribonuclease, while for residues 32 and 34 it is approximately 7-11 times greater. CD analysis of RNase A and RNase B revealed the carbohydrate moiety to have a small stabilizing effect (approximately 5 kJ/mol) on the protein.

Capillary Electrophoretic Separation of Human Recombinant Erythropoietin (r-HuEPO) Glycoforms

Free zone capillary electrophoresis was investigated for the separation of glycoforms in recombinant erythropoietin (r-HuEPO). Reduction of electroendoosmotic flow through the addition of 1,4-diaminobutane and in the presence of urea resulted in optimum separation of all of the major glycoforms. Incubation of r-HuEPO with neuraminidase caused the slowest migrating species to diminish first in response, indicating that these contained the highest numbers of sialic acids. Additional evidence was obtained from spiking r-HuEPO with individual glycoforms isolated by preparative isoelectric focusing. These results indicate that the separation occurs in a predictable manner in order of increasing number of sialic acids contained in the glycoforms. As a consequence, the electropherograms provide the same information as that obtained from conventional gel isoelectric focusing, although separating by a different mechanism.

In vivo clearance of tissue plasminogen activator: The complex role of sites of glycosylation and level of sialylation

The involvement of a high mannose oligosaccharide in the clearance of tissue plasminogen activator (t-PA) has been reported previously. Removal of this oligosaccharide by mutation of amino acid #117 from asparagine (Asn) to glutamine (Gln) generates a t-PA variant designated LAt-PA which was shown to clear significantly more slowly than the native molecule. Here we report clearance studies in rabbits with LAt-PA produced in mouse C127 cells under differing growth conditions which indicate that the half-life of the variant is additionally influenced by the degree of sialylation of the remaining complex oligosaccharides. The data suggest that, in addition to the mannose receptor, the asialoglycoprotein receptor is also involved in the hepatic clearance of LAt-PA from the circulation. In addition, t-PA has been shown to exist in two major glycoforms termed Type I and Type II, which are defined by the presence or absence of a complex oligosaccharide on Asn 184 respectively. We also separated the individual glycoforms of LAt-PA and determined their monosaccharide composition. The glycoform with a complex oligosaccharide at Asn 184 has an extended half-life in rabbits compared to the glycoform lacking this oligosaccharide.

HPLC of individual site glycoforms in serum orosomucoid

The glycoforms contained within each glycosylation site of orosomucoid can be separated by reverse-phase HPLC without removal of the associated peptide or chemical derivatization. The chromatographic properties of the glycopeptides are dominated by the peptide portion but modified by the hydrophilicity of the glycan in a regular manner depending on its size. Assignments for known structures are readily made, permitting quantitative assessment of changes in glycoform concentration at individual glycosylation sites.