ABSTRACT
Occurrence of the adhesion/growth-regulatory galectins as family sets the challenge to achieve a complete network analysis. Along this route taken for a well-suited model organism (chicken), we fill the remaining gap to characterize its seventh member known from rat as galectin-related inter-fiber protein (GRIFIN) in the lens. Its single-copy gene is common to vertebrates, with one or more deviations from the so-called signature sequence for ligand (lactose) contact. The chicken protein is a homodimeric agglutinin with capacity to bind ß-galactosides, especially the histo-blood group B tetrasaccharide, shown by solid-phase/cell assays and a glycan microarray. Mass spectrometric identification of two lactose-binding peptides after tryptic on-bead fragmentation suggests an interaction at the canonical region despite a sequence change from Arg to Val at the site, which impairs reactivity of human galectin-1. RT-PCR and Western blot analyses of specimen from adult chicken organs reveal restriction of expression to the lens, here immunohistochemically throughout its main body. This report sets the stage for detailed structure-activity studies to define factors relevant for affinity beyond the signature sequence and to perform the first complete network analysis of the galectin family in developing and adult organs of a vertebrate.
Subject(s)
Avian Proteins/genetics , Eye Proteins/genetics , Galectins/genetics , Gene Expression Profiling/methods , Gene Regulatory Networks , Amino Acid Sequence , Animals , Avian Proteins/chemistry , Avian Proteins/metabolism , Binding Sites/genetics , Blotting, Western , Chickens , Eye Proteins/classification , Eye Proteins/metabolism , Galectins/classification , Galectins/metabolism , Humans , Immunohistochemistry , Lactose/metabolism , Lens, Crystalline/metabolism , Phylogeny , Protein Binding , Protein Multimerization , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis. In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.
Subject(s)
Histocompatibility Antigens Class I/metabolism , Immunoglobulin G/metabolism , Mass Spectrometry/methods , Oxidation-Reduction , Receptors, Fc/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Chromatography, Liquid , Humans , Immunoglobulin G/chemistry , Peptide Mapping , Surface Plasmon ResonanceABSTRACT
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.