Breaking the Light and Heavy Chain Linkage of Human Immunoglobulin G1 (IgG1) by Radical Reactions

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2011 May 25

PMID 21606498

Citations 7

Authors

Boxu Yan

Daniel Boyd

Affiliations

Soon will be listed here.

Abstract

We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys(215) of the light chain and Cys(225) of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C(α)ONH-) and the side chain of Cys allows an electron transfer to the α carbon, forming a new intermediate radical species (-(·)C(α)(O(-))NH-) at Cys(225). Dissociative deamidation occurs along the N-C(α) bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.

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References

Davies M, Donkor R, Dunster C, Gee C, Jonas S, Willson R . Desferrioxamine (Desferal) and superoxide free radicals. Formation of an enzyme-damaging nitroxide. Biochem J. 1987; 246(3):725-9. PMC: 1148337. DOI: 10.1042/bj2460725. View

Wentworth Jr P, Jones L, Wentworth A, Zhu X, Larsen N, Wilson I . Antibody catalysis of the oxidation of water. Science. 2001; 293(5536):1806-11. DOI: 10.1126/science.1062722. View

Yan B, Yates Z, Balland A, Kleemann G . Human IgG1 hinge fragmentation as the result of H2O2-mediated radical cleavage. J Biol Chem. 2009; 284(51):35390-402. PMC: 2790968. DOI: 10.1074/jbc.M109.064147. View

Nathan C . Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J Clin Invest. 2003; 111(6):769-78. PMC: 153776. DOI: 10.1172/JCI18174. View

Poole L, Claiborne A . The non-flavin redox center of the streptococcal NADH peroxidase. I. Thiol reactivity and redox behavior in the presence of urea. J Biol Chem. 1989; 264(21):12322-9. View

Chance B, Sies H, Boveris A . Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979; 59(3):527-605. DOI: 10.1152/physrev.1979.59.3.527. View

Ouellette D, Alessandri L, Piparia R, Aikhoje A, Chin A, Radziejewski C . Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine. Anal Biochem. 2009; 389(2):107-17. DOI: 10.1016/j.ab.2009.03.027. View

Dillon T, Bondarenko P, Rehder D, Pipes G, Kleemann G, Ricci M . Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. J Chromatogr A. 2006; 1120(1-2):112-20. DOI: 10.1016/j.chroma.2006.01.016. View

Gray H, Winkler J . Electron tunneling through proteins. Q Rev Biophys. 2004; 36(3):341-72. DOI: 10.1017/s0033583503003913. View

10.

Cohen S, Price C, Vlasak J . Beta-elimination and peptide bond hydrolysis: two distinct mechanisms of human IgG1 hinge fragmentation upon storage. J Am Chem Soc. 2007; 129(22):6976-7. DOI: 10.1021/ja0705994. View

11.

Wang P, Sanders P . Immunoglobulin light chains generate hydrogen peroxide. J Am Soc Nephrol. 2007; 18(4):1239-45. DOI: 10.1681/ASN.2006111299. View

12.

Boveris A, Chance B . The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J. 1973; 134(3):707-16. PMC: 1177867. DOI: 10.1042/bj1340707. View

13.

Davies M, Forni L, Shuter S . Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals. Chem Biol Interact. 1987; 61(2):177-88. DOI: 10.1016/0009-2797(87)90038-x. View

14.

Imlay J . Pathways of oxidative damage. Annu Rev Microbiol. 2003; 57:395-418. DOI: 10.1146/annurev.micro.57.030502.090938. View

15.

Jacob C, Knight I, Winyard P . Aspects of the biological redox chemistry of cysteine: from simple redox responses to sophisticated signalling pathways. Biol Chem. 2006; 387(10-11):1385-97. DOI: 10.1515/BC.2006.174. View

16.

Schubert J, Sharma V, White E, Bergelson L . Catalytic decomposition of hydrogen peroxide by copper chelates and mixed ligand complexes of histamine in the presence of phosphate buffer in the neutral pH region. J Am Chem Soc. 1968; 90(16):4476-8. DOI: 10.1021/ja01018a061. View

17.

Carter P . Potent antibody therapeutics by design. Nat Rev Immunol. 2006; 6(5):343-57. DOI: 10.1038/nri1837. View

18.

Takamoto K, Chance M . Radiolytic protein footprinting with mass spectrometry to probe the structure of macromolecular complexes. Annu Rev Biophys Biomol Struct. 2006; 35:251-76. DOI: 10.1146/annurev.biophys.35.040405.102050. View

19.

Yates Z, Gunasekaran K, Zhou H, Hu Z, Liu Z, Ketchem R . Histidine residue mediates radical-induced hinge cleavage of human IgG1. J Biol Chem. 2010; 285(24):18662-71. PMC: 2881791. DOI: 10.1074/jbc.M110.108597. View

20.

SHIMAZU F, Tappel A . SELENOAMINO ACIDS: DECREASE OF RADIATION DAMAGE TO AMINO ACIDS AND PROTEINS. Science. 1964; 143(3604):369-71. DOI: 10.1126/science.143.3604.369. View