Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Abstract

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. 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.Walsh C.T. Posttranslational Modification of Proteins: Expanding Nature's Inventory. 1st Ed. Roberts and Co. Publishers, Greenwood Village, CO2006Google Scholar). Although the impact of post-translational modifications (PTMs) 1The abbreviations used are:PTMpost-translational modificationDSCdifferential scanning calorimetryH/DXhydrogen/deuterium exchangeIgG1immunoglobulin γ1UPLCultraperformance LC. on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2.Johnson L.N. Barford D. The effects of phosphorylation on the structure and function of proteins.Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 199-232Crossref PubMed Scopus (222) Google Scholar), 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. post-translational modification differential scanning calorimetry hydrogen/deuterium exchange immunoglobulin γ1 ultraperformance LC. Many biopharmaceutical companies are pursuing antibody drugs (3.Carter P.J. Potent antibody therapeutics by design.Nat. Rev. Immunol. 2006; 6: 343-357Crossref PubMed Scopus (906) Google Scholar). 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.Saphire E.O. Parren P.W. Pantophlet R. Zwick M.B. Morris G.M. Rudd P.M. Dwek R.A. Stanfield R.L. Burton D.R. Wilson I.A. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.Science. 2001; 293: 1155-1159Crossref PubMed Scopus (768) Google Scholar). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5.Garber E. Demarest S.J. A broad range of Fab stabilities within a host of therapeutic IgGs.Biochem. Biophys. Res. Commun. 2007; 355: 751-757Crossref PubMed Scopus (196) Google Scholar). 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.Jefferis R. Glycosylation as a strategy to improve antibody-based therapeutics.Nat. Rev. Drug Discov. 2009; 8: 226-234Crossref PubMed Scopus (661) Google Scholar). As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7.Walsh G. Jefferis R. Post-translational modifications in the context of therapeutic proteins.Nat. Biotechnol. 2006; 24: 1241-1252Crossref PubMed Scopus (747) Google Scholar). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood a functional In the of IgG1s, with and a mass a large of PTMs can be both in vivo cellular and in (as a of and that and monitored PTMs on IgG1s methionine oxidation, and or of and C.T. the of Ed. PubMed Scopus Google Scholar). of these modifications a of the protein and may not can have impact on the function, and biological of a protein that can as well as with other proteins R. PubMed Scopus Google Scholar). The PTMs can IgG1 in or have no the of PTMs, the of the and understanding the structural effects of PTMs are important of protein commonly IgG1 modifications are methionine oxidation and of which has to biological function (6.Jefferis R. Glycosylation as a strategy to improve antibody-based therapeutics.Nat. Rev. Drug Discov. 2009; 8: 226-234Crossref PubMed Scopus (661) Google R. of methionine oxidation on the binding of human IgG1 to Fc and Fc Immunol. 2009; PubMed Scopus Google Scholar). oxidation has in protein and oxidation have to an of and of Biophys. Res. 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