The low affinity IgG receptor FcγRIIB contributes to the binding of the mast cell specific antibody, mAb BGD6

Michel F. Guiraldelli, Elsa H. Berenstein, Ana Grodzki, Reuben P. Siraganian, Maria Celia Jamur, Constance Oliver

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of ∼110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to FcγRIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab′)2 fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to FcγRIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab′)2 fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have FcγRIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a ∼110 kDa protein and the Fc tail interacts with FcγRIIB (CD32).

Original languageEnglish (US)
Pages (from-to)2411-2418
Number of pages8
JournalMolecular Immunology
Volume45
Issue number8
DOIs
StatePublished - Apr 1 2008
Externally publishedYes

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Keywords

  • CD32
  • Expression cloning
  • Mast cells
  • Precursor

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

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