Document Detail


Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon Beta-1a: potential implications for protein aggregation and immunogenicity.
MedLine Citation:
PMID:  23534382     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.
Authors:
Riccardo Torosantucci; Victor S Sharov; Miranda van Beers; Vera Brinks; Christian Schöneich; Wim Jiskoot
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-05-02
Journal Detail:
Title:  Molecular pharmaceutics     Volume:  10     ISSN:  1543-8392     ISO Abbreviation:  Mol. Pharm.     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-06-03     Completed Date:  2014-01-15     Revised Date:  2014-06-04    
Medline Journal Info:
Nlm Unique ID:  101197791     Medline TA:  Mol Pharm     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2311-22     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Ascorbic Acid / chemistry
Benzene Derivatives / chemistry
Chromatography, Gel
Circular Dichroism
Copper / chemistry
Humans
Interferon-beta / chemistry*,  immunology
Methylamines / chemistry
Molecular Structure
Spectrometry, Fluorescence
Spectrometry, Mass, Electrospray Ionization
Tandem Mass Spectrometry
Grant Support
ID/Acronym/Agency:
P01 AG012993/AG/NIA NIH HHS; P01AG12993/AG/NIA NIH HHS
Chemical
Reg. No./Substance:
0/4-(aminomethyl)benzene sulfonate; 0/Benzene Derivatives; 0/Methylamines; 77238-31-4/Interferon-beta; 789U1901C5/Copper; PQ6CK8PD0R/Ascorbic Acid
Comments/Corrections

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