Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function

Sarah E. Henson, Donald Smith, Susan A. Boackle, V. Michael Holers, David R. Karp

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)97-109
Number of pages13
JournalJournal of Immunological Methods
Volume258
Issue number1-2
DOIs
StatePublished - Dec 1 2001

Fingerprint

Biotinylation
B-Lymphocytes
Complement 3d Receptors
Ligands
Complement Receptors
Complement C3
B-Lymphoid Precursor Cells
Streptavidin
Complement Activation
Cell Surface Receptors
B-Cell Lymphoma
Protein Sorting Signals
complement C3d,g
Antigen-Antibody Complex
Fluorescent Dyes
Mitogens
Flow Cytometry
Phosphotransferases
Complementary DNA
Monoclonal Antibodies

Keywords

  • B lymphocytes
  • Biotin
  • Complement
  • Flow cytometry
  • Tetramer

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function. / Henson, Sarah E.; Smith, Donald; Boackle, Susan A.; Holers, V. Michael; Karp, David R.

In: Journal of Immunological Methods, Vol. 258, No. 1-2, 01.12.2001, p. 97-109.

Research output: Contribution to journalArticle

Henson, Sarah E. ; Smith, Donald ; Boackle, Susan A. ; Holers, V. Michael ; Karp, David R. / Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function. In: Journal of Immunological Methods. 2001 ; Vol. 258, No. 1-2. pp. 97-109.
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