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     

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.     

Effect of aminoacylation on tRNA conformation

Translational diffusion coefficients have been simulated for various conformations of tRNA^Phe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes.     

De novo generation of a PrPSc-like conformation in living cells

Conformational conversion of the cellular PrPC protein to PrPSc is a central aspect of the prion diseases, but how PrP initially converts to this conformation remains a mystery. Here we show that PrP expressed in the yeast cytoplasm, instead of the endoplasmic reticulum, acquires the characteristics of PrPSc, namely detergent insolubility and a distinct pattern of protease resistance. Neuroblastoma cells cultured under reducing, glycosylation-inhibiting conditions produce PrP with the same characteristics. We therefore describe what is, to our knowledge, the first conversion of full-length PrP in a heterologous system, show the importance of reducing and deglycosylation conditions in PrP conformational transitions, and suggest a model for initiating events in sporadic and inherited prion diseases.     

Antibody crystallization on phospholipid films: dynamics and the effects of antibody conformation

Monoclonal antibodies from two-dimensional (2-D) crystals when bound to haptenated phospholipid monolayers in physiological conditions and at ambient temperatures. IgG1 forms two crystal phases: a linear strand phase and a high-order hexagonal phase. The relative distribution of these two phases is dependent on temperature, pH, and salt concentration. This dependence is one that is associated with protein intramolecular interactions rather than lipid-lipid or lipid-protein interactions for a number of reasons: 1) Polyclonal antibodies against the hapten DNP do not organize into any crystal structure for any of the experimental conditions used. 2) Slightly denatured IgG (through storage at 4 degrees C, for example) does not readily crystallize and a shift in the temperature dependence for forming the hexagonal phase is observed. 3) There is no pH driven transition in crystallization tendency for IgE anti-DNP but a transition to disorder is observed at above 30 degrees C. No such transition exists for IgG1. Observation of the dynamics of crystal growth shows a clear and marked dependence on pH and temperature that is in accord with the results of long-term incubations. It is found that high pH retards crystal growth very significantly for IgG1 but not for IgE. Also, the crystal growth rate of 4 degrees C-stored IgG1 is greatly reduced over fresh IgG1 (-80 degrees C stored). Furthermore, it is found that the linear phase of IgG1 is an extremely rapidly forming phase but one that is metastable against the hexagonal phase.     

Effect of peptide conformation on membrane permeability.

The effect of peptide conformational constraint on the peptide permeation across the model membranes was examined by determining the permeability of pairs of cyclic and acyclic peptides related to c[d-Pen2, d-Pen5] enkephalin (DPDPE). The peptides were cyclized by formation of an intramolecular disulfide bridge between the second and fifth residues composed of either d-penicillamine or cysteine. In each case the acyclic peptide was three to seven times more permeable than corresponding cyclic peptide. The possibility that the differences in permeability of cyclic and acyclic peptides is based on the greater conformational freedom of the acyclic peptides in the presence of membrane was examined in more detail by isothermal titration calorimetric studies of Trp6-DPDPE and its acyclic analog. The membrane binding of the acyclic peptide is a more exothermic process than binding of its cyclic Trp6-DPDPE. The transfer of acyclic peptide from water to membrane is an enthalpy driven process, whereas the transfer of the cyclic peptide is driven by entropy.     

Effect of G-1 on histidine tRNA microhelix conformation

Histidine tRNAs (tRNA^His) are unique in that they possess an extra 5′-base (G-1) not found in other tRNAs. Deletion of G-1 results in at least a 250-fold reduction in the rate of histidine charging in vitro. To better understand the role of the G-1 nucleotide in defining the structure of tRNA^His, and to correlate structure with cognate amino acid charging, NMR and molecular dynamics (MD) studies were performed on the wild-type and a ΔG-1 mutant Escherichia coli histidine tRNA acceptor stem microhelix. Using NMR-derived distance restraints, global structural characteristics are described and interpreted to rationalize experimental observations with respect to aminoacylation activity. The quality of the NMR-derived solution conformations of the wild-type and ΔG-1 histidine microhelices (micro helix^His) is assessed using a variety of MD-based computational protocols. Most of the duplex regions of the acceptor stem and the UUCG tetraloop are well defined and effectively superimposable for the wild-type and ΔG-1 mutant microhelix^His. Differences, however, are observed at the end of the helix and in the single-stranded CCCA-3′ tail. The wild-type microhelix^His structure is more well defined than the mutant and folds into a ‘stacked fold-back’ conformation. In contrast, we observe fraying of the first two base pairs and looping back of the single-stranded region in the ΔG-1 mutant resulting in a much less well defined conformation. Thus the role of the extra G-1 base of the unique G-1:C73 base pair in tRNA^His may be to prevent end-fraying and stabilize the stacked fold-back conformation of the CCCA-3′ region.     

Sequencing antibody repertoires: The next generation

Genomic studies have been revolutionized by the use of next generation sequencing (NGS), which delivers huge amounts of sequence information in a short span of time. The number of applications for NGS is rapidly expanding and significantly transforming many areas of life sciences. The field of antibody research and discovery is no exception. Several recent studies have harnessed the power of NGS for analyzing natural or synthetic immunoglobulin repertoires with unprecedented resolution and exploring alternative paths for antibody discovery. Thus, appreciating and then exploiting these advances is essential for staying at the edge of antibody innovation.Key words: next generation sequencing, phage display, hybridoma, antibody discovery, in vitro selection, immunization     

Anti-insulin antibody structure and conformation. I. Molecular modeling and mechanics of an insulin antibody.

A knowledge-based three-dimensional model of an anti-insulin antibody, 125, was constructed using the structures of conserved residues found in other known crystallographic immunoglobulins. Molecular modeling and mechanics were done with the 125 amino acid sequences using QUANTA and CHARMm on a Silicon Graphics 4D70GT workstation. A minimal model was made by scaffolding using crystallography coordinates of the antibody HyHEL-5, because it had the highest amino acid sequence homology with 125 (84% light chain, 65% heavy chain). The three hypervariable loop turns that are longer in 125 than in HyHEL-5 (L1, L3, and H3) were modeled separately and incorporated into the HyHEL-5 structure; then other amino acid substitutions were made and torsions optimized. The 125 model maintains all the structural attributes of an antibody and the structures conserved in known antibodies. Although there are many polar amino acids (especially serines) in this site, the overall van der Waals surface shape is determined by positions of aromatic side chains. Based on this model, it is suggested that hydrogen bonding may be key in the interaction between the human insulin A chain loop antigenic epitope and 125.     

Synthetic approach to the generation of antibody diversity

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]