Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

OBJECTIVE: To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). METHODS: A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. RESULTS: DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. CONCLUSION: Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Eukaryotic initiation factor 4A is the component that interacts with ATP in protein chain initiation.

Protein synthesis in a resolved homogenate of wheat germ requires ATP and eight factors functioning at the level of protein chain initiation. To identify the component(s) interacting with ATP, the different factors were treated with the ATP affinity analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and tested for their function in protein synthesis. The activity of eukaryotic initiation factor 4A (eIF4A) was strongly curtailed, whereas all other factors were unaffected. At a concentration of 250 microM, AMP, ADP, and ATP protected eIF4A against FSBA inactivation, whereas at a concentration 50 microM, protection was afforded only by ATP. GTP did not protect at a concentration of 250 microM. In another approach, the substrate analogue 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) was found to inhibit protein synthesis in a manner, at least in part, competitive with ATP. Supplementing a TNP-ATP inhibited reaction with eIF4A substantially reversed the inhibition. Except for a small effect by factor C1, no reversal was obtained with any other component. Finally, a preincubation of ribosomes with ATP, mRNA, and eIF4A resulted in the formation of a complex capable of TNP-ATP-resistant amino acid incorporation. These data are interpreted to indicate that the primary interaction of ATP is with eIF4A. A model is proposed reconciling this conclusion with other observations relevant to the mRNA . ribosome attachment reaction.