The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biologic activity and protein conformation.

Russell, John; Katzhendler, Jehoshua; Kowalski, Kazimierz; Schneider, Arthur B.; Sherwood, Louis M.

doi:10.1016/s0021-9258(19)70135-5

Cited by 6 publications

(2 citation statements)

References 19 publications

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“…For example, Trp can be oxidized to N -formyl kynurenine (NFK) and kynurenine and sensitize the reduction of peptide and/or disulfide bonds, forming thiyl radicals and thiolate. , The latter process requires photoinduced electron or H atom transfer between Trp and the disulfide bond. Modification of Trp residues in proteins has resulted in conformational changes and loss of biologic activity and presents a major concern for the production and formulation of biopharmaceuticals. − Moreover, kynurenines are efficient photosensitizers, inferring that Trp oxidation can lead to additional photosensitivity of a protein formulation …”

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confidence: 99%

Light-Induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

et al. 2013

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The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by αC-βC cleavage of the Trp side chain. The resulting glycyl radicals are either reduced to Gly, or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photo-induced electron- or hydrogen-transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals.

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confidence: 99%

Light-Induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

et al. 2013

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“…Single amino acid changes in proteins have been studied through chemical modification or amino acid substitution to identify the residues responsible for binding activity and structure changes of proteins. − It has been shown that modification of a single amino acid residue can affect the stability, structure, and activity of proteins. For instance, chemical oxidation of a single Trp in human placental lactogen resulted in conformational changes and reduced lactogenic activity . A single Trp-139 in the thermostable d -amino acid transaminase was critical for its activity, as proved by site-directed mutagenesis .…”

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Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus

Wei¹,

Feng²,

Lin³

et al. 2007

Anal. Chem.

152

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We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.

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