Protein posttranslational modification

Many polypeptides undergo covalent modification after (or sometimes during) their ribosomal assembly. The most commonly observed such PTMs are listed in Table 2.7. Such modifications generally influence either the biological activity or the structural stability of the polypeptide. The majority of therapeutic proteins bear some form of PTM. Although glycosylation represents the most common such modification, additional PTMs important in a biopharmaceutical context include carboxylation, hydroxylation, sulfation and amidation; these PTMs are now considered further.

2.5.1 Glycosylation

Glycosylation (the attachment of carbohydrates) is one of the most common forms of PTM associated with eukaryotic proteins in general, particularly eukaryotic extracellular and cell surface proteins. In the case of some glycoproteins, removal of the sugar component has no detectable effect upon the biological properties (deglycosylated forms of the glycoprotein can be generated by including inhibitors of the glycosylation pathway, e.g. the antibiotic tunicamycin, in the cell growth media,

Table 2.7 Types of PTM that polypeptides may undergo. Refer to text for additional details



Proteolytic processing Glycosylation


Acetylation Acylation




Y-Carboxyglutamate formation

ADP-ribosylation Disulfide bond formation

Various proteins become biologically active only upon their proteolytic cleavage (e.g. some blood factors)

For some proteins glycosylation can increase solubility, influence biological half-life and/or biological activity Influences/regulates biological activity of various polypeptide hormones Function unclear

May help some polypeptides interact with/anchor in biological membranes Influences biological activity/stability of some polypeptides

Influences biological activity of some neuropeptides and the proteolytic processing of some polypeptides Important to the structural assembly of certain proteins

Important in allowing some blood proteins to bind calcium

Regulates biological activity of various proteins Helps stabilize conformation of some proteins or by enzymatic degradation of the glycocomponent of preformed glycoproteins using glucosidase enzymes). However, in other cases the sugar component plays a direct role in the biological activity of the glycoprotein (Table 2.8). Native hCG (Chapter 11), for example, is a heavily glycosylated gonadotrophic hormone. Removal of its sugar components usually abolishes its ability to induce a biological response, although the hormone's binding affinity for its receptor remains unaltered, or is sometimes actually increased. Therapeutic proteins now approved for general medical use that are glycosylated are listed in Table 2.9.

Carbohydrate side chains are synthesized by a family of enzymes known as glycosyltransferases, located mainly in the endoplasmic reticulum. Two types of glycosylation can occur: N-linked and O-linked. In the case of N-linked glycosylation, the sugar chain (the oligosaccharide) is attached to the protein via the nitrogen atom of an asparagine (Asn) residue, whereas in O-linked systems the sugar chain is attached to the oxygen atom of hydroxyl groups, usually those of serine or thero-nine residues (Figure 2.9). Monosaccharides most commonly found in the sugar side chain(s) include mannose, galactose, glucose, fucose, ^-acetylgalactosamine, N-acetylglucosamine, xylose and sialic acid. These can be joined together in various sequences and by a variety of glycosidic linkages. The carbohydrate chemistry of glycoproteins, therefore, is quite complex. The structure of two such example oligosaccharide chains is presented in Figure 2.10.

N-linked glycosylation is sequence specific, involving the transfer of a pre-synthesized oligosaccharide chain to an asn residue found in a characteristic sequence Asn-X-Ser, or Asn-X-Thr or Asn-X-Cys, where X represents any amino acid residue, with the exception of proline. An additional glycosylation determinant must also apply, as not all potential N-linked sites are glycosylated in some proteins. The pre-synthesized oligosaccharide side chain then undergoes

Table 2.8 The potential roles and effects of the glycocomponent of glycoproteins. Reproduced from Walsh, G. and Jefferies, R. (2006). Nature Biotechnology 24, 1241-1252



Protein folding

Glycosylation can effect local protein secondary

structure and help direct folding of the polypeptide


Protein targeting/trafficking

The glycocomponent can participate in the sorting/

directing of a protein to its final destination

Ligand recognition/binding

The carbohydrate content of antibodies, for example,

plays a role in antibody binding to monocyte

Fc receptors and interaction with complement

component C1q

Biological activity

The carbohydrate side chain of gonadotrophins is

essential to the activation of gonadotrophin signal



Sugar side chains can potentially stabilize a

glycoprotein in a number of ways, including

enhancing its solubility, shielding hydrophobic

patches on its surface, protection from proteolysis

and by direct participation in intrachain stabilizing


Regulates protein half-life

High levels of sialic acid (a family of acidic sugars

that often caps sugar side chains) can increase

a glycoprotein's plasma half-life. Exposure of

galactose residues can decrease plasma half-life by

promoting uptake via hepatic galactose residues.

Yeast glycosylation is of a 'high mannose' type,

which can also drive rapid removal from circulation

via specific cell-surface mannose receptors


Some glycosylation motifs characteristic of plant-

derived glycoproteins (often containing fucose

and xylose residues) are highly immunogenic in


additional glycosyltransferase-mediated trimming/modification. The determinants of O-linked glycosylation are even less well understood. Characteristic sequence recognition is not apparent in most cases, and three-dimensional structural features may be more important in such instances. Some glycosylated proteins will be characterized by one or more N-linked sugar side chains, others by one or more O-linked side chains, and still others by both N- and O-linked chains. Human EPO (Chapter 10), for example, displays three N-linked and one O-linked sugar side chain.

For any glycoprotein, the exact composition and structure of the carbohydrate side chain can vary slightly from one molecule of that glycoprotein to the next. This results in microheterogeneity which can be directly visualized by analytical techniques such as isoelectric focusing (Chapter 7). Also contributing to heterogeneity can be variable site glycosylation, in which some glycosylation sites remain unoccupied within a proportion of the glycoprotein molecules. The overall basis of heterogeneity is likely due to factors such as glycosyltransferase substrate specificity and the fact

Table 2.9 Approved therapeutic proteins (listed by trade name) that are glycosylated. These products are discussed in subsequent chapters. Reproduced from Walsh, G. and Jefferies, R. 2006. Nature Biotechnology 24, 1241-1252

Product category

Specific products (by trade name)

Blood factors, anticoagulants and thrombolytics



EPO and colony-stimulating factors



Activase, Advate, Benefix, Bioclate, Helixate/ Kogenate, Metalyse/TNKase, Novoseven, Recombinate, Refacto, Xigiris Avastin, Bexxar, Erbitux, Herceptin, Humaspect, Humira, Mabcampath/Campath-H1, Mabthera/ Rituxan, Mylotarg, Neutrospec, Oncoscint, Orthoclone OKT-3, Prostascint, Raptiva, Remicade, Simulect, Synagis, Xolair, Zenapax, Zevalin Gonal F, Luveris, Ovitrelle/Ovidrel, Puregon/

Follistim, Thyrogen Epogen/Procrit, Leukine, Neorecormon, Nespo/Aranesp Avonex, Rebif

Aldurazyme, Amevive, Cerezyme, Enbrel, Fabrazyme, Inductos, Infuse, Osigraft/OP-1 implant, Pulmozyme, Regranex, Replagal

ch2 I

chain |



Serine — sidechain



Figure 2.9 (a) N-linked versus (b) O-linked glycosylation. 'Sugar' represents an oligosaccharide chain, an example of which is provided in Figure 2.10

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: W-acetyl glucosamine; GalNAc: W-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-y (IFN-y), 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 significance, if any, of altered glycosylation patterns/microheterogeneity is not always predictable 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

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






Figure 2.11 y-Carboxylation of glutamate residues (Glu) yields y-carboxyglutamate (Gla), whereas P-hy-droxylation of aspartate (Asp) yields P-hydroxyaspartate (Hya) and P-hydroxylation of asparagine (Asn) yields P-hydroxyasparagine (Hyn)

2.5.3 Sulfation and amidation

Sulfation and amidation are two additional PTMs characteristic of a small number of biopharma-ceuticals. Sulfation entails the enzyme-catalysed attachment of sulfate (SO4^) groups to target polypeptides, usually via specific tyrosine side chains. Sulfation often plays a role in protein-protein interactions, and lack of sulfation tends to reduce a polypeptide's activity, as opposed to abolishing it completely. Notable therapeutic proteins that are sulfated in their natural state include the anticoagulant hirudin (Chapter 12) and blood factors VIII and IX. The recombinant forms of these proteins produced by genetic engineering generally have reduced levels/absence of sulfation, but yet they remain therapeutically effective.

Amidation refers to the replacement of a protein's C-terminal carboxyl group with an amide group (COOH ^ CONH2). This PTM is usually characteristic of peptides (very short chains of amino acids), as opposed to the longer polypeptides, but one therapeutic polypeptide (salmon calcitonin, Chapter 11) is amidated, and amidation is required for full functional activity. Overall, the function(s) of amidation is not well understood, although in some cases at least it appears to contribute to peptide/polypeptide stability and/or activity.

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  • Marco
    How amino acids are removed in post translational modification?
    8 years ago

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