Protein structure - Pharmaceutical Biotechnology: Concepts and Aapplications - page 35

SA Gal Glc NAc Man

Man Glc NAc Glc NAc Asn

SA Gal Glc NAc Man

Gal Glc NAc Ser

SA

Figure 2.10 Structure of two sample oligosaccharide side chains (one N-linked the other O-linked) found in
glycoproteins. Man: manose; Gal: galactose; SA: sailic acid; GlcNAc: N -acetyl glucosamine; GalNAc: N -acetyl
galactosamine

that a proportion of the glycosylated proteins may be exported from the cell before they are fully
processed by glycosyltransferases.
Virtually all therapeutic glycoproteins, even when produced naturally in the body, exhibit such
heterogeneity; for example, two species of human interferon-γ (IFN-γ), one of molecular mass 20
kDa and the other of 25 kDa, differ from each other only in the degree and sites of (N-linked)
glycosylation.
Furthermore, the glycosylation patterns obtained when human glycoproteins are expressed
in non-human eukaryotic expression systems (e.g. animal cell culture) are usually somewhat
different from the glycosylation pattern associated with the native human protein. The
glycosylation pattern of human tPA produced in transgenic animals, for example, is different to
the pattern obtained when the same gene is expressed in a recombinant mouse cell line. Both
these patterns are, in turn, different to the native human pattern. The clinical signifi cance, if
any, of altered glycosylation patterns/microheterogeneity is not always predic table and is best
determined by direct clinical trials. If the product is found to be safe and effective, then routine
end-product quality control (QC) analysis for carbohydrate-based microheterogeneity is carried
out more to determine batch-to-batch consistency (which is desirable) rather than to detect
microheterogeneity per se .

2.5.2 Carboxylation and hydroxylation

-hydroxylation are PTMs characteristic of a limited number of proteins,
mainly a subset of proteins that function in the haemostatic process. γ-Carboxylation entails the
enzymatic conversion of the side chains of specifi c glutamate residues in target proteins, forming
γ-carboxyglutamate (conversion of 'Glu' residues to 'Gla' residues; Figure 2.11a). β-Hydroxyla-
tion usually entails the hydroxylation of target aspartate (Asp) residues yielding

γ -Carboxylation and β

-hydroxyaspar-
tate (Asp → Hya; Figure 2.11b). Both PTMs help mediate the binding of calcium ions, which is
important/essential to the effective functioning of blood factors VII, IX and X, as well as activated
protein C and protein S of the anticoagulant system (Chapter 12).

β