The crystal structure of rabbit IgG-Fc

We report the structure of the Fc fragment of rabbit IgG at 1.95 A (1 A=0.1 nm) resolution. Rabbit IgG was the molecule for which Porter established the four-chain, Upsilon-shaped structure of the antibody molecule, and crystals of the Fc ('Fragment crystallisable') were first reported almost 50 years ago in this journal [Porter, R. R. (1959) Biochem. J. 73, 119-126]. This high-resolution analysis, apparently of the same crystal form, reveals several features of IgG-Fc structure that have not previously been described. More of the lower hinge region is visible in this structure than in others, demonstrating not only the acute bend in the IgG molecule that this region can mediate, as seen in receptor complexes, but also that this region has a tendency to adopt a bent structure even in the absence of receptor. As observed in other IgG-Fc structures, the Cgamma2 domains display greater mobility/disorder within the crystals than the Cgamma3 domains; unexpectedly the structure reveals partial cleavage of both Cgamma2 intra-domain disulphide bonds, whereas an alternative conformation for one of the cysteine residues in the intact bridge within the more ordered Cgamma3 domains is observed. The N-linked oligosaccharide chains at Asn(297) are well-defined and reveal two alternative conformations for the galactose units on each of the alpha(1-6)-linked branches. The presence of this galactose unit is important for stabilizing the structure of the entire branched carbohydrate chain, and its absence correlates with the severity of autoimmune conditions such as rheumatoid arthritis in both human clinical studies and in a rabbit model of the disease. Rabbit IgG, through this high-resolution structure of its Fc region, thus continues to offer new insights into antibody structure.     

Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement.

The recognition that certain biological effector functions associated with the Fc region of human IgG are mediated exclusively by either the Cgamma2 or Cgamma3 domains prompted a study of some of the physical properties of the isolated domains in an attempt to correlate these with functional differentiation. The degree of aromatic chromophore exposure of intact Fc and fragments corresponding to the Cgamma2 and Cgamma3 domains were determined by solvent perturbation difference spectroscopy using 20% ethylene glycol. For the monomeric Cgamma2 fragment one of the two tryptophans and all four of the tyrosines were exposed to solvent. In the pFc' fragment, which represented a dimer of two intact Cgamma3 domains, an average of 0.4 of the two tryptophans of 3.3 of the five tyrosines per chain were exposed. These data were consistent with the suggested involvement of tryptophan in complement fixation since Cgamma2 binds C1q but pFc' does not. Several fragments derived from the Cgamma3 region had previously been shown to have differing environments for their aromatic side chains from circular dichroism studies. These fragments have now been shown to exhibit different degrees of chromophore exposure to solvent. Removal of the carboxy-termimal heptapeptide from the intact, Cgamma3 domain resulted in a fragment not only showing a greater exposure of aromatic residues but also having the ability to bind Clq. Our data suggest that the structural requirements for C1Q binding may be quite commonplace within Fc, but tertiary folding limits their expression except in Cgamma2 in the native molecule. The solvent perturbation observed with Fc was somewhat lower than would have been expected from the results with the isolated domains, suggesting that interdomain interactions may result in burial of aromatic residues.     

Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments

Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and β1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.     

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.     

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety,functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as nextgeneration therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.     

Structure of the Murine Unglycosylated IgG1 Fc Fragment

A prototypic IgG antibody can be divided into two major structural units: the antigen-binding fragment (Fab) and the Fc fragment that mediates effector functions. The IgG Fc fragment is a homodimer of the two C-terminal domains (C_H2 and C_H3) of the heavy chains. Characteristic of the Fc part is the presence of a sugar moiety at the inner face of the C_H2 domains. The structure of this complex branched oligosaccharide is generally resolved in crystal structures of Fc fragments due to numerous well-defined sugar-protein interactions and a small number of sugar-sugar interactions. This suggested that sugars play an important role in the structure of the Fc fragment. To address this question directly, we determined the crystal structure of the unglycosylated Fc fragment of the murine IgG1 MAK33. The structures of the C_H3 domains of the unglycosylated Fc fragment superimpose perfectly with the structure of the isolated MAK33 C_H3 domain. The unglycosylated C_H2 domains, in contrast, approach each other much more closely compared to known structures of partly deglycosylated Fc fragments with rigid-body motions between 10 and 14 Å, leading to a strongly “closed” conformation of the unglycosylated Fc fragment. The glycosylation sites in the C′E loop and the BC and FG loops are well defined in the unglycosylated C_H2 domain, however, with increased mobility and with a significant displacement of about 4.9 Å for the unglycosylated Asn residue compared to the glycosylated structure. Thus, glycosylation both stabilizes the C′E-loop conformation within the C_H2 domain and also helps to ensure an “open” conformation, as seen upon Fc receptor binding. These structural data provide a rationale for the observation that deglycosylation of antibodies often compromises their ability to bind and activate Fcγ receptors.     

The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein.

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.     

Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding

Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.     

Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.The structure of many proteins can be altered by post-translational modifications (1). Although the impact of post-translational modifications (PTMs)¹ on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2), it is less defined for other PTMs and in many cases is protein-dependent. Because there are many important downstream effects of PTMs, including changes in protein localization, protein and cellular diversification, protein functionality, protein stability, protein life cycle, and so forth, understanding how PTMs alter protein structure for as many proteins as possible in a timely manner is a highly desirable goal. Furthermore, in an age where recombinant proteins are being used to treat disease, it becomes ever more important to understand how particular modifications may alter the structure and eventually the function of therapeutic proteins. To realize these goals, methods that permit access to conformational information for modified forms of therapeutic proteins must be developed and refined. In this report, we will illustrate how MS can contribute to structural proteomics by describing our recent work with a recombinant monoclonal antibody (an IgG1), which represents an important class of therapeutic proteins.Many biopharmaceutical companies are pursuing antibody drugs (3). In particular, the IgG1 subclass of antibodies has evolved into a commonly used therapeutic option for the treatment of a wide range of diseases. IgG1s consist of a dimer of identical heavy chains and light chains that fold to form (from N to C terminus) the variable, CL, CH1, CH2, and CH3 domains (as an example, see Ref. 4). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5). The variable, CL, and CH1 domains are collectively referred to as the Fab (fragment antigen binding) portion of IgG1, which is responsible for recognizing a specific antigen. The CH2 and CH3 domains together are referred to as the Fc (fragment crystallizable) portion, which carries out effector functions such as binding to Fcγ receptors. These effector functions are essential to many therapeutic antibodies, especially when antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity are involved in the mechanisms of action (6).As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood at a functional level. In the case of IgG1s, with over 1300 amino acid residues and a molecular mass approaching 150 kDa, a large array of PTMs can be incorporated both in vivo (during cellular synthesis) and in vitro (as a result of handling and processing steps that occur during purification, vialing, and storage). Commonly monitored PTMs on IgG1s include methionine oxidation, asparagine and glutamine deamidation, N-terminal acetylation or cyclization, glycation of lysine, and variable glycosylation (8). Some of these modifications affect only a small percentage of the protein product, and their presence may not change overall outcome. Others, however, can have significant impact on the structure, function, and biological activities of a protein that can involve self-association as well as interactions with other proteins (9). The same PTMs can affect different IgG1 molecules in different ways or have no effect(s) at all. Therefore assessing the presence of PTMs, determining the relative level of the modifications, and understanding the structural effects of PTMs are all important during development of protein biopharmaceuticals.Two commonly studied IgG1 modifications are methionine oxidation and glycosylation, each of which has been shown to affect biological function (6, 10). Methionine oxidation has been implicated in protein stability (inducing aggregation), and increased oxidation levels have been shown to provoke an immunogenic response (11–13). Elevated levels of methionine oxidation in an IgG1 were shown to impact neonatal Fc receptor (FcRn) and protein A binding (10). Variable glycosylation (i.e. different levels of sialic acid, galactose, fucose, or high mannose structures) is known to influence thermal stability and effector functions (14–16). Previous studies have shown that removal of fucose from the glycan present on the Fc portion of an IgG1 can greatly enhance Fc binding to FcγRIIIa, but removal of the entire glycan nearly abolishes FcγRIIIa binding (17). As oxidation and changes to the glycan are both common IgG1 modifications, we were interested in determining the conformational effects of oxidation, afucosylation, and galactosylation and correlating any conformational changes that were observed with changes of FcγRIIIa binding activity.Conformational analysis of large proteins like antibodies, however, is not trivial. Traditional biophysical techniques such as circular dichroism, DSC, and fluorescence provide useful information, but these techniques look at the entire protein and provide only a global view (18). NMR and x-ray crystallography can both provide high resolution structural analysis, but each is faced with limitations that often make the study of an intact IgG1 difficult or nearly impossible (19–21). Recently we described how hydrogen/deuterium exchange (H/DX) MS could be used to study the conformation and conformational dynamics of an intact IgG1 with resolution down to stretches of several amino acid residues (22). For the present work, we used H/DX MS to study the impact of galactosylation, oxidation, and afucosylation on the conformation and dynamics of an intact IgG1. We also studied the complex of IgG1 and FcγRIIIa to map the points of interaction and probe any changes in the dynamics of the IgG1 as a result of FcγRIIIa interaction. Finally, we correlated the functional activity of all the proteins that were studied by H/DX MS with the observed conformational disturbance(s). Such correlations are important to connect structure with function and to understand whether a particular PTM is something that may affect the therapeutic value of a recombinant protein.     

Conformational studies of the glycopeptide Ac-Tyr-[Man5GlcNAc-beta-(1-->4)GlcNAc-beta-(1-->Ndelta)]-Asn-Leu-Thr-Se r-OBz and the constituent peptide and oligosaccharide

Glycopeptides of desired structure can be conveniently prepared by the coupling of reducing oligosaccharides to aspartic acid of peptides via their glycosylamines formed in the presence of saturated aqueous ammonium hydrogen carbonate. The resulting oligosaccharide chains are N-linked to asparagine as in natural glycoproteins, allowing different peptide oligosaccharide combinations to be analysed for conformational effects. In the present paper, a pentapeptide of ovalbumin was coupled to Man5GlcNAc2 oligosaccharide and the glycopeptide and the two parent compounds compared by NMR ROESY experiments and molecular dynamics simulations. Despite the small size of the peptide, conformational effects were observed suggestive of the oligosaccharide stabilising the peptide in solution and of the peptide influencing oligosaccharide conformation. These effects are relevant to the function of glycosylation and the enzymic processing of oligosaccharide chains.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH2 domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH₂ domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.Key words: monoclonal antibody, glycosylation, stability, liquid chromatography-mass spectroscopy, Fourier transform infrared, fluorescence spectroscopy, size-exclusion chromatography, differential scanning calorimetry     

Oxidative iodination of rabbit IgG: localization of markers in an Fc-fragment and effects of modification]

A comparative study of rabbit IgG, both native and modified ones, designed to assess the functional activity of these proteins under oxidative iodination conditions has been carried out. Polyclonal IgG, its antigen-specific fraction and Iodogen as an oxidant were used. Polyclonal antibodies directed against the CH2 domain of IgG, protein A targeted at the CH-2-CH3 domain interface and ferritin testing the conformation of the antigen-binding Fv fragment, were applied as conformational probes for assessing the changes in the IgG conformation. By taking advantage of pepsin proteolysis of [125I]-IgG, from 80% to 92% of the label was found to be localized within the CH3 domain, thus implying the domain-selective nature of iodination, when the degree of modification was below 0.1 atom of iodine per IgG molecule. Yet, when the three above-mentioned conformational probes were used, considerable alterations in the conformation of not only the CH2 domain and CH2-CH3 domain interface, but in the Fv domain being a part of the Fab fragment, were observed. By using competitive enzyme immunoassay for the straightforward comparative evaluation of functional properties of "cold" (native) and 125I-modified IgG, the deleterious effect of the oxidant (Iodogen) rather than iodine atom substitution at the phenolic ring of Tyr residues was shown to be the major determinant of alterations in the IgG molecule.     

Conformational analysis of blood group A-active glycosphingolipids using HSEA-calculations. The possible significance of the core oligosaccharide chain for the presentation and recognition of the A-determinant

Conformational analysis of four different A-active glycosphingolipids, A types 1-4, was carried out using HSEA-calculations with the GESA-program. In their minimum energy conformations the oligosaccharide chains are more or less curved; in particular the type 3 and 4 have a strongly bent shape. When the carbohydrate structures are linked to ceramide, using the conformational features predominantly observed in crystal structures of membrane lipids, rather drastic differences in the orientation of the oligosaccharide chains are obtained. For the type 1 glycosphingolipid the model study indicates that the A-determinant extends almost perpendicularly to the membrane plane whereas for type 2, 3 and 4 the terminal part of the oligosaccharide chains is more parallel to the membrane. The fucose branch on type 3 and type 4 thereby appears directed towards the environment whereas for type 2 it would face the membrane. Due to restrictions imposed by the membrane layer this core specific orientation is largely preserved even if the flexibility of the saccharide-ceramide linkage is taken into account. Hydrophilic and hydrophobic sites on the surface of the different oligosaccharide chains in their minimum energy conformation were located using the GRID-program. It is suggested that the core-dependent presentation of the A-determinant might explain the chain type specificity observed for different monoclonal anti-A antibodies. The results further suggest that assay systems ensuring a membrane-like presentation of the glycolipid antigen should be used in studies of glycolipid/protein interactions.     

Folding mechanism of the CH2 antibody domain

The immunoglobulin C(H)2 domain is a simple model system suitable for the study of the folding of all-beta-proteins. Its structure consists of two beta-sheets forming a greek-key beta-barrel, which is stabilized by an internal disulfide bridge located in the hydrophobic core. Crystal structures of various antibodies suggest that the C(H)2 domains of the two heavy chains interact with their sugar moieties and form a homodimer. Here, we show that the isolated, unglycosylated C(H)2 domain is a monomeric protein. Equilibrium unfolding was a two-state process, and the conformational stability is remarkably low compared to other antibody domains. Folding kinetics of C(H)2 were found to consist of several phases. The reactions could be mapped to three parallel pathways, two of which are generated by prolyl isomerizations in the unfolded state. The slowest folding reaction, which was observed only after long-term denaturation, could be catalyzed by a prolyl isomerase. The majority of the unfolded molecules, however, folded more rapidly, on a time-scale of minutes. Presumably, these molecules also have to undergo prolyl isomerization before reaching the native state. In addition, we detected a small number of fast-folding molecules in which all proline residues appear to be in the correct conformation. On both prolyl isomerization limited pathways, the formation of partly structured intermediates could be observed.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Inhibition of processing of asparagine-linked carbohydrate chains on IgG2a by using swainsonine has no influence upon antibody effector functions in vitro

Removal of asparagine (Asn)-linked carbohydrate chains from IgG antibody molecules reduces their antibody effector functions such as C activation and FcR binding. We have prepared IgG2a mAb with modified structure of carbohydrate chains by treating the hybridoma cells with swainsonine, which inhibits the processing of Asn-linked carbohydrate chains at the site of action of mannosidase II. These antibodies have obtained the capacity to bind lentil lectin and have become sensitive to endoglycosidase H digestion, indicating the structural changes of oligosaccharides from complex type to hybrid type. They behaved in an identical manner to the normal IgG2a antibodies with regards to extracellular secretion, Ag-binding capacity, C-mediated hemolysis and FcR-mediated functions. Critical moieties of Asn-linked carbohydrate chains on IgG molecules to retain their antibody effector functions were discussed.     

Structural study of the sugar moieties of monoclonal antibodies secreted by human-mouse hybridoma

Six monoclonal antibodies, three each of human IgG1 and IgG2 subclasses, were obtained from human-mouse hybridomas. Structural study of their asparagine-linked sugar chains was performed to elucidate the regulatory mechanism of secreted monoclonal IgG glycosylation. The sugar moieties were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by NaB3H4 reduction after N-acetylation. Structural study of each oligosaccharide by lectin affinity column chromatography, sequential exoglycosidase digestion, and methylation analysis indicated that almost all of them were biantennary complex-type sugar chains containing Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)GlcNAc as core structures. Bisecting N-acetylglucosamine residue, which is present in human IgG but not in mouse IgG, could not be detected at all. The molar ratio of each oligosaccharide from the six IgG samples was different. However, no subclass specificity was detected except that all IgG1 contained neutral, mono-, and disialylated sugar chains, whereas IgG2 did not contain disialylated ones. The molar ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid was also different for each IgG. All six IgGs contained monoantennary complex-type and high mannose-type oligosaccharides which had never been detected in serum IgGs of various mammals so far investigated. These results indicated that the processing of asparagine-linked sugar chains of IgG is less complete in human-mouse hybridoma than in human or mouse B cells, and that the glycosylation machinery of the mouse cells is dominant in the hybrid cells.     

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.