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Protein Scaffolds for Multivalent Display of High‐Affinity CD22 Ligands
Author(s) -
O’Reilly Mary K.,
Liao Liang,
Rillahan Cory,
Collins Brian E.,
Han Shoufa,
Kitov Pavel,
Bundle David R.,
Paulson James C.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.810.1
Subject(s) - cd22 , chemistry , ligand (biochemistry) , glycan , lectin , flow cytometry , biochemistry , microbiology and biotechnology , receptor , glycoprotein , cell , biology , cd19
CD22 is a sialic acid‐binding immunoglobulin‐like lectin (Siglec) that is expressed on the surface of B cells and functions as a regulator of B cell receptor signaling. The relationship between glycan binding and immunomodulatory function is being explored, and has led to the development of ligand‐based probes of CD22. CD22 binds to glycan ligands bearing the terminal sequence NeuAcα2‐6Gal with low affinity (K d = ~0.2 mM), requiring multivalency to achieve stable binding. We have used a decavalent anti‐nitrophenol (NP) IgM antibody to display hetero‐bi‐functional ligands comprising a CD22 ligand coupled to the antigen NP. In this approach the protein scaffold participates in ligand‐driven assembly of multivalent IgM‐CD22 complexes, which can be demonstrated on immobilized recombinant CD22 by ELISA or flow cytometry of CD22‐immobilized magnetic beads. More importantly, assembly is observed on the surface of native B cells, as detected by immunofluorescence and flow cytometry, even though the ligand‐binding site of CD22 is largely masked by endogenous glycan ligands on the cell surface. This assembly is sensitive to the length and structure of the spacer, and also to the valency of the scaffold, as anti‐NP IgA and IgG display decreased avidity compared to anti‐NP IgM. The observation that a divalent protein scaffold succeeds where synthetic probes of higher valency do not validates this approach.