ABSTRACT
Hydrogen/deuterium exchange mass spectrometry (HDX MS) was used in two case studies to evaluate the impact of methionine (Met) oxidation on the biological functions of IgG1 antibodies. In the first case study, linear correlations were observed between the oxidation of the conserved Fc methionine residues and the loss of neonatal Fc receptor (FcRn) binding and complement-dependent cytotoxicity (CDC) activity. Both heavy chain (HC) residues Met257 and Met433 were located near the FcRn binding interface as indicated by HDX MS and structural modeling; however, HC Met257 oxidation was further demonstrated to have a more significant impact on FcRn binding than HC Met433 oxidation. In addition, oxidation of HC Met257 and HC Met433 could disrupt protein conformation at the CH2-CH3 interface and prevent IgG oligomerization, which is needed for C1q binding and subsequent CDC activity. In the second case study, HDX MS demonstrated that oxidation of the two complementary determining region (CDR) methionine residues had little or no impact on antigen binding of the antibody. Together, these results suggested that HDX MS is a powerful tool for evaluating the impact of individual post translational modifications (PTMs) on the biological activities of antibodies, even when the PTM levels are relatively low. The high selectivity and sensitivity of this method makes it a valuable tool for assisting the critical quality attributes (CQAs) assessment of antibodies.
Subject(s)
Antibodies, Monoclonal/metabolism , Histocompatibility Antigens Class I/metabolism , Immunoglobulin G/metabolism , Methionine/chemistry , Receptors, Fc/metabolism , Antibodies, Monoclonal/chemistry , Deuterium , Histocompatibility Antigens Class I/chemistry , Humans , Immunoglobulin G/chemistry , Mass Spectrometry/methods , Oxidation-Reduction , Protein Binding , Protein Conformation , Protein Processing, Post-Translational , Receptors, Fc/chemistryABSTRACT
Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT-PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.
Subject(s)
DNA Methylation , Esophagus/metabolism , Gene Expression Regulation , Homeodomain Proteins/genetics , Promoter Regions, Genetic , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Bile Acids and Salts/pharmacology , CDX2 Transcription Factor , DNA Primers , Decitabine , Epithelial Cells/metabolism , Esophagus/cytology , Humans , Immunohistochemistry , Molecular Sequence Data , Polymerase Chain Reaction , TransfectionABSTRACT
PURPOSE: Cyclic AMP response element binding protein binding protein (CBP), a nuclear transcriptional coactivator protein, is an important component of the cAMP signal transduction pathway. In this study, we systematically analyzed the pattern and frequency of CBP gene alterations in esophageal squamous cell carcinoma (ESCC) samples from Linzhou (Linxian), China. EXPERIMENTAL DESIGN: Using microsatellite markers D16S475, D16S2622, and D16S523 within the chromosome 16p13.3 locus flanking the CBP gene, we observed loss of heterozygosity (LOH), microsatellite instability (MSI), or homozygous deletion in 16 of 26 ESCC samples. Additional ESCC samples were analyzed using different sets of microsatellite markers (CS1-CS5) within the introns or in close proximity to the 3' end of the CBP gene. RESULTS: The data showed that CBP gene LOH or MSI occurred in 9 of 19 ESCC samples. A detailed genetic alteration map of the CBP gene showed that an LOH or MSI hot spot occurred within intron 2 of the CBP gene. Furthermore, ESCC samples were investigated for CBP gene mutation by conformation sensitive gel electrophoresis and DNA sequencing. These results revealed that most of the shifted fragments contained internal tandem duplication (ITD), frequently in the regions encoding the histone acetyltransferase domain and COOH-terminal transactivating domain one of the CBP gene. The presence of ITD within the CBP gene was additionally confirmed by Southern blot analysis and sequencing. CONCLUSIONS: These studies show that LOH and ITD of the CBP gene are frequent genetic events in human ESCC. These alterations may have functional importance in the development of human ESCC.
