Using phage display for rational engineering of a higher-affinity humanized 3' phosphohistidine-specific antibody

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Histidine phosphorylation is an understudied post-translational modification (PTM) with 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis) isoforms.
• The development of specific reagents, particularly high-affinity antibodies, is limited due to the labile phosphoramidate (P-N) bond of phosphohistidine.

Purpose of the Study:

• To engineer high-affinity, specific antibodies for detecting native 3-phosphohistidine (3-pHis) targets.
• To provide novel tools for studying the biological roles of 3-pHis in mammalian cells.

Main Methods:

• Humanization of a rabbit anti-3-phosphotazettine (3-pTza) antibody (SC44-8) into a phage display scaffold (hSC44).
• Screening of six Fab phage-displayed libraries to select antibodies with enhanced affinity and specificity for 3-pHis over 3-pTza.
• Structural and quantum mechanical analyses to elucidate the molecular basis of antibody-antigen interactions.

Main Results:

• Engineered antibody hSC44.20N32F^L demonstrated approximately 10-fold higher affinity for 3-pHis compared to the parental hSC44.
• Structural studies provided the first atomic resolution of antibody-pHis peptide complexes, revealing mechanisms for 3-pHis/3-pTza discrimination.
• The novel antibodies successfully detected 3-pHis in mammalian cells via immunoblotting and immunofluorescence staining.

Conclusions:

• A generalizable strategy for engineering antibodies against labile post-translational modifications was established.
• Novel, high-affinity 3-pHis-specific antibodies were developed, facilitating research into the cellular functions of 3-pHis.
• The findings open new avenues for exploring the significance of histidine phosphorylation in cell biology.