Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function.
CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.
Henson
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SE
The Harold C. Simmons Arthritis Research Center and the Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA.
Smith
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D
Boackle
S A
SA
Holers
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VM
Karp
D R
DR
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D001710:Biotin; D002460:Cell Line; D003179:Complement C3b / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D020836:Protein Structure, Quaternary; D017464:Receptors, Complement 3d / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D018076:DNA, Complementary; D008024:Ligands; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins; C045477:complement C3d,g; D001710:Biotin; D003179:Complement C3b