The two membrane isoforms of human IgE assemble into functionally distinct B cell antigen receptors

Abstract

The human C epsilon gene expresses two membrane IgE heavy chain mRNAs which differ in the sequence that encodes their extracellular membrane-proximal domain. In the long IgE isoform (mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant (mSIgE). We have now generated B cell transfectoma cell lines that express these two isoforms and show that both types of mIgE form functional B cell antigen receptors (BCR). Both receptors associate with the Ig-alpha/Ig-beta heterodimer, as well as with protein kinases that are capable of phosphorylating this complex. Upon their cross-linking, both receptors can activate protein tyrosine kinases that phosphorylate the same substrate proteins. Both IgE receptors also associate with two novel proteins that do not bind to mIgM. Apart from these similarities, the two IgE-BCRs show several differences of which some are analogous to the differences between the IgM- and IgD-BCRs. First, the mSIgE is transported to the cell surface at a higher rate than the mLIgE. Second, the two IgE-BCRs associate with differently glycosylated Ig-alpha proteins, the mLIgE associates with the completely glycosylated form, whereas the mSIgE associates with an Ig-alpha glycoform that is partially sensitive to endoglycosidase H. Third, the kinetics of protein tyrosine phosphorylation induced by receptor cross-linking is significantly different for the two IgE-BCRs. Finally, cross-linking of the mSIgE-BCR leads to growth inhibition of the B cell transfectoma, whereas signaling through the mLIgE-BCR does not affect the cellular proliferation. These data show that the two human membrane IgE isoforms assemble into functionally distinct antigen receptors which can induce different cellular responses.

Figure 1

Schematic representation of the two membrane IgE H chain isoforms. (a) Diagram of the 3′ part of the Cε and the pattern of alternative splicing that generates the εCH4-M1-M2 and εCH4-M1′-M2 membrane transcripts. Open boxes represent coding sequences and shadowed boxes represent 3′ untranslated regions; stop codons are indicated by asterisks. (b) Amino acid sequence of the COOH terminus of the short (εCH4-M1-M2) and long (εCH4-M1′-M2) ε chains. The extra 52 amino acids present in the extracellular membrane–proximal domain of the long ε chain are underlined. (c) Diagram of the chimeric mouse V_H-NIP/Cε membrane gene constructs. The 3′ ends of the two membrane ε isoforms were cloned in their cDNA form (starting from the CH3 exon) into pCIG-Cε, using the indicated restriction enzyme sites.

Figure 2

Assembly and transport of m_LIgE and m_SIgE. WEHI-m_SIgE, WEHI-m_LIgE, and wild-type WEHI cells were pulse labeled with [³⁵S]methionine for 15 min and chased for the indicated times in the presence of excess cold methionine. Cellular extracts were immunoprecipitated with the indicated antibodies and (a) analyzed on a nonreducing 6% SDS-PAGE or (b) treated with Endo H and separated on reducing 10% SDS-PAGE.

Figure 3

Immunoprecipitation of BCRs from biotin-labeled cell-surface proteins. WEHI-m_SIgE and WEHI-m_LIgE were surface biotinylated and treated with digitonin lysis buffer to preserve the BCR complexes. (a) Lysates were treated first with anti-IgE, and then with anti-IgM sera to immunoprecipitate the IgE-BCR and endogenous IgM-BCR complexes, respectively. Immunoprecipitated material was treated with Endo H or PNGase as indicated, and analyzed by reducing 10% SDS-PAGE. Arrowheads indicate the position of the Ig-α polypeptide after removal of one of the two N-linked carbohydrate moieties by Endo H. Ig-α(N₋₂) and Ig-β(N₋₃) correspond to the Ig-α and Ig-β polypeptides after removal by PNGase of the two or three N-linked carbohydrate moieties, respectively. (b) Reprecipitation of the Ig-α/Ig-β heterodimer dissociated by NP-40 from the m_SIgE-BCR and m_LIgEBCR. The immunoprecipitation was done with an anti Ig-α serum and analyzed on a 10% SDS-PAGE under reducing conditions.

Figure 4

Bidimensional analysis of surface-biotinylated IgEBCRs. Anti-IgE immunoprecipitates from the two transfectomas were analyzed by bidimensional (nonreducing/reducing) PAGE. The migration of the same material run only under reducing conditions or nonreducing conditions is shown in the left lane of each gel, or as a separate lane on top of each gel, respectively. Arrowheads indicate the position of the dissociated εBAP37 and εBAP41 proteins. The other components of the IgE-BCRs are indicated in the figure.

Figure 5

In vitro phosphorylation of BCR associated proteins. IgE- and IgM-BCR from the WEHI-m_SIgE and WEHI-m_LIgE transfectomas were immunoprecipitated, incubated with [γ³²P]ATP, and analyzed on reducing 10% SDS-PAGE. Treatment with Endo H or PNGase is indicated on top of each gel. Arrowheads, Ig-α(N₋₂), and Ig-β(N₋₃) as in legend to Fig. 3.

Figure 6

Quantitation of m_SIgE- and m_LIgE-BCR levels on the surface of transfected WEHI cells. The level of surface IgE was determined in each cell line by fluorescent flow cytometry analysis with a rabbit anti– human IgE antibody and FITCconjugated swine anti–rabbit IgG.

Figure 7

Kinetics of PTK substrate phosphorylation upon cross-linking of the m_SIgE-, m_LIgE-, and IgM-BCR. Wildtype WEHI, WEHI-m_LIgE, or WEHI-m_SIgE cells were incubated for various times with antiIgE or anti-IgM antibodies, as indicated on top of each gel. Cellular extracts were then prepared and tyrosine phosphorylated proteins detected by immunoblotting with an anti-phosphotyrosine mAb.

Figure 8

Proliferation of WEHI-m_LIgE and WEHI-m_SIgE cells after cross-linking of mIgE. 2 × 10⁴ cells were incubated with 0, 1, 5, 25, or 50 μg/ml of polyclonal anti–human IgE anti-serum. Results show one representative of three separate experiments with similar results. Each point represents an average of triplicate cultures (SEM <5%).