CH1-CH2 Domains Research Articles

Characterization and applications of nanobodies against Pseudomonas aeruginosa Exotoxin A selected from single alpaca B cells

We have established a highly efficient 96-well format based strategy to characterize nanobody (Nb) from an expressed alpaca Nb repertoire by combining heavy-chain antibody variable region gene cloning with Nb expression and reactivity profiling at the single cell level. Individual alpaca B lymphocytes were isolated based on B cell surface marker and membrane type antigen specific antibodies expression by fluorescence activated cell sorting (FACS). Single cell reverse transcription and polymerase chain reaction (PCR) amplification of CH1 and CH2 domain enable the identification and exclusion of B cell clones that express regular antibodies. The corresponding heavy-chain antibody variable region gene transcripts, which are CH1-CH2 domain negative, are then amplified from single B cell clones by nested PCR. Then Nbs were expressed in Escherichia coli cells and four Nbs showed high affinity against Pseudomonas aeruginosa Exotoxin A (PEA). Moreover, two PEA Nbs that recognize unique epitopes on PEA have been successfully applied to protect human cells from PEA induced apoptosis. In summary, our FACS and single cell reverse transcription polymerase chain reaction (RT-PCR) based strategy for identifying recombinant Nbs from single alpaca B cells allows highly efficient and unbiased characterization of the expressed Nb repertoires by sequence analysis, expression and parallel antibody reactivity testing.

Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1–CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1–CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1–CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1–CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1–CH2 affinity is both increased and decreased by modifications that change the effective “openness” of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution “openness,” suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1–CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1–CH2 binding properties.

Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS.

Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal downstream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL-CH1 and CH1-CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL-CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites.

Smooth muscle α-actinin binds tightly to fesselin and attenuates its activity toward actin polymerization

Fesselin is an actin binding protein from smooth muscle that nucleates actin polymerization in a Ca(++)-calmodulin dependent manner, bundles actin and inhibits the actin-activated ATPase activity of myosin S1. We now report that fesselin binds to smooth muscle alpha-actinin. Binding was measured by blot overlay, affinity chromatography and sedimentation methods. Binding was moderate with an association constant of 1-4 x 10(7) M(-1) assuming a 1:1 association of fesselin with alpha-actinin. Fesselin binds to the central spectrin domain repeat region of alpha-actinin but not to the CH1-CH2 domain. Fesselin accelerates the polymerization of actin. This activity of fesselin was attenuated by alpha-actinin. These observations support the role of fesselin in organizing the cytoskeleton.

Nucleotide sequence divergence of mouse immunoglobulin gamma 1 and gamma 2b chain genes and the hypothesis of intervening sequence-mediated domain transfer.

The nucleotide sequences of the constant portions of mouse immunoglobulin gamma 1 and gamma 2b genes were compared. A remarkable homology was found in a long (about 500 nucleotides) continuous segment including the entire CH1 coding region and about the first half of the first intervening sequence. Furthermore, comparison of amino acid sequences of four gamma-class chains revealed that the CH1 domain shows limited divergence among gamma 1, gamma 2a, and gamma 2b. Interestingly, the homology region extends to the CH2 domain in gamma 2a and gamma 2b. These findings suggest that, during their evolution, a double unequal crossing-over event has taken place at different intervening sequences, resulting in the transfer of the DNA segment coding for the CH1 domain or CH1-CH2 domains. A possible evolutionary implication for such an "intervening sequence-mediated domain transfer" event is discussed.