The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the NADPH oxidase complex, a multicomponent enzyme system that initiates a cascade of reactive oxygen species that play a critical role in innate immunity and vascular physiology. Epitope-mapped, monoclonal antibodies (mAb) that recognize the large (gp91phox) and small (p22phox) subunits of Cyt b provide valuable reagents that have been used to examine structural and mechanistic aspects of oxidase function. In the present study, the heavy and light chain variable region genes of the Cyt b-specific mAbs 44.1, NS5, and NL7 have been amplified by RT-PCR, cloned and subject to DNA sequence analysis. Since the 5′ degenerate primer sets used for mAb gene amplification were observed to introduce extensive heterogeneity into the heavy and light chain FR1 regions, N-terminal protein sequence analysis was also conducted to obtain the correct amino acid sequence of this region. In order to confirm the identity of the cloned genes, intact mAbs were resolved by two-dimensional electrophoresis and subject to in-gel tryptic digestion for analysis by both MALDI and nanospray LC-MS/MS. Databases searches using the derived mAb sequences predicted residues comprising CDR loops, identified candidate germline genes, and showed the respective germline genes to accurately predict the N-terminal amino acid residues for each variable region. The above studies report the amino acid sequence of Cyt b-specific mAb variable region genes with high confidence and provide essential information for future efforts at Cyt b structure analysis by resonance energy transfer and X-ray crystallography.
- Flavocytochrome b
- Mass spectrometry