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Endosomal Targeting by the Cytoplasmic Tail of Membrane Immunoglobulin

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Science  18 Apr 1997:
Vol. 276, Issue 5311, pp. 407-409
DOI: 10.1126/science.276.5311.407

Abstract

Membrane-bound immunoglobulin (mIg) of the IgG, IgA, and IgE classes have conserved cytoplasmic tails. To investigate the function of these tails, a B cell line was transfected with truncated or mutated γ2a heavy chains. Transport to the endosomal compartment of antigen bound by the B cell antigen receptor did not occur in the absence of the cytoplasmic tail; and one or two mutations, respectively, in the Tyr-X-X-Met motif of the tail partially or completely interrupted the process. Experiments with chimeric antigen receptors confirmed these findings. Thus, a role for the cytoplasmic tail of mIg heavy chains in endosomal targeting of antigen is revealed.

The B cell antigen receptor (BCR) is a multiprotein complex that includes the membrane-bound immunoglobulin molecule (mIg) and the Ig-α,Ig-β heterodimer (1). The latter molecules function as the signaling subunit of the BCR. They are also required for the intracellular transport of IgM-BCR to the endosomal compartment, where the bound antigen is proteolytically degraded (2). All classes of mIg are associated with the Ig-α,Ig-β heterodimer (3), but the heavy chains differ in the length of their cytoplasmic tails: there are 3 amino acids for μm and δm tails and 28 amino acids for γm and ɛm tails. No function has so far been attributed to the conserved cytoplasmic sequence of mIgG molecules that are expressed on memory B cells.

To analyze the function of the 28–amino acid cytoplasmic tail of the γ2am heavy chain, we truncated or mutated the sequence (4) coding for this tail in the expression vector pSV2neoγ2am (5). The chain lacking all cytoplasmic amino acids except for the three KVK (6) residues (which are identical to the COOH-terminus of the μm chain) we called γ2amtl. Point mutations were introduced to change the YXXM motif in the γ2am cytoplasmic sequence to either LXXM (γ2amY20L) or LXXL (γ2amY20L,M23L). Expression vectors for these heavy chains were transfected into K46λ12 B lymphoma cells expressing a λ1 light chain. The expressed wild-type and mutated γ2am chains associate with the λ1 light chain to form 5-iodo-4-hydroxy-3-nitrophenyl-acetyl (NIP)–specific mIgG2a molecules.

After surface biotinylation of K46λγ2am and K46λγ2amtl cells, the wild-type and tailless IgG2a-BCR complexes were affinity-purified over NIP-Sepharose (7) and analyzed by protein immunoblotting (Fig. 1). This analysis confirmed that the γ2amtl chain has a lower molecular weight than the wild-type γ2am chain (Fig. 1, lanes 4 and 2) and showed that both mIgG2a molecules are associated with the Ig-α,Ig-β heterodimer to the same extent. Yet unidentified surface proteins of 41 and 42 kD were copurified together with the wild-type but not truncated IgG2a-BCR complex. These molecules may thus require the γ2am tail for efficient binding. A fluorescence-activated cell sorter analysis confirmed that similar amounts of mIgG2atl and wild-type mIgG2a were expressed on K46 cells, whereas the two point-mutated mIgG2a molecules were expressed in amounts that were reduced by a factor of 3 to 5.

Figure 1

Composition of wild-type IgG2a-BCR (lane 2) and tailless IgG2atl-BCR (lane 4) on the surface of K46λ12 cells. After surface biotinylation, a 1% digitonin lysate of the different γ2am transfectants was incubated with either Sepharose (S; lanes 1 and 3) or NIP-Sepharose (NP; lanes 2 and 4), and bound molecules were analyzed by SDS-PAGE on 10% gels with protein immunoblotting.

The endosomal transport of antigen bound to wild-type or mutated IgG2a-BCR was tested in an ovalbumin (OVA) peptide presentation assay (8). The different γ2am transfectants of K46λ12 cells were cocultured with the T helper cell line 3DO54.8, which is specific for the OVA 323-339 peptide in the presence of NIP-OVA or OVA alone (Fig. 2). The K46λγ2am cells, which express wild-type IgG2a-BCR, were able to present the antigenic peptide to the T cells when exposed to low amounts of NIP-OVA, whereas exposure to the same amount of OVA did not result in antigen presentation (Fig.2B; P < 0.001). K46λγ2amtl cells, which express the tailless IgG2atl-BCR complex, did not present the OVA peptide even when cultured with large amounts of the specific antigen (Fig. 2C). The same defect was found in two independent γ2am transfectants of K46λ12 that expressed an IgG2a-BCR with a double (Y → L, M → L) (6) mutation of the YXXM motif. These are referred to as K46λγ2amY20L,M23L (Fig. 2, E and F). K46λγ2amY20L cells, expressing an IgG2a-BCR with a single Y → L mutation of the YXXM motif, had a modest but not statistically significant capacity to present antigen (Fig. 2D; P < 0.3). The endosomal targeting function of the γ2am tail was tested by a second approach that used expression vectors for chimeric single-chain antigen receptors (scAgR) (9). These receptors consist of the covalently linked VH and VL domains of the NIP-specific antibody B1-8 (10) attached to the CD8α hinge region and the transmembrane part of the T cell receptor ζ chain, to which different tail sequences can be appended. K46 cells were transfected with several variants of scAgR: one with a wild-type γ2am tail, one with a YXXM point mutation, and one with a truncated (tl) scAgR, which carried RRIDP (6) as a cytoplasmic tail. These were tested in the antigen presentation assay. After exposure to the antigen NIP-OVA, the K46scγ2am cells efficiently presented the OVA peptide (Fig. 3A; P < 0.001), whereas K46scγ2amY20L,M23L (Fig. 3C) or K46sctl (Fig. 3D) cells did not. The K46scγ2amY20L cells, which express a chimeric receptor with the Y → L point mutation of the YXXM motif, were able to present antigen, although they did so less efficiently than K46scγ2am cells (Fig. 3B; P < 0.01).

Figure 2

Dose response of antigen presentation by (A) untransfected K46λ12 cells and the different γ2am-transfectants, (B) K46λγ2am, (C) K46λγ2amtl, (D) K46λγ2amY20L, (E) K46λγ2amY20L,M23L#1, and (F) K46λγ2amY20L,M23L#2 exposed for 24 hours to different concentrations of either NIP-OVA (black squares) or OVA (white triangles). Mean values and standard deviations from triplicates are shown. IL-2 production is presented as relative OD450, as described in (8).

Figure 3

Dose response of antigen presentation by K46 cells expressing different chimeric scAgRs. The transfectants (A) K46scγ2am, (B) K46scγ2amY20L, (C) K46scγ2amY20L,M23L, (D) K46sctl, (E) K46scIg-α, and (F) K46scIg-αM1 were exposed for 24 hours to different concentrations of either NIP-OVA (black squares) or OVA (white triangles).

It has previously been shown that chimeric receptor molecules containing the cytoplasmic component of Ig-β or Ig-α can mediate transport to the antigen-processing compartment (2, 11). We have generated transfectants of K46 expressing scAgR with either the wild-type or an altered Ig-α tail sequence carrying a Y → F (M1) mutation (12) of both tyrosines of the immunoreceptor tyrosine-based activation motif (ITAM). Upon exposure to low doses of NIP-OVA, both transfectants were able to efficiently present the OVA peptide (Fig. 3, E and F; P < 0.001). These results showed that the Ig-α tail has a targeting function that is not abolished after a Y → F mutation of the two tyrosines of the ITAM.

The aggregation of chimeric receptors that contain a γ2am tail does not result in intracellular signaling (2, 13), which indicates that the internalization and presentation of antigen are independent from signal transduction. The sequence around the YXXM motif (SPDYRNMIG) (6) is conserved in all IgG classes and has similarities to the NPXY (6) internalization signal of the low-density lipoprotein receptor for which a type I β-turn structure has been demonstrated (14). Such a structure seems to mediate the contact between the receptor and intracellular transporter molecules. The tail of the ɛm chain carries, at the same position as the YXXM motif, a YXXI sequence that may function as an endosomal transport signal for the mIgE molecule.

The targeting function of the YXXM motif is abolished by the double mutation (Y → L, M → L) but not by a single Y → L mutation. Apparently, a bulky hydrophobic amino acid such as leucine can functionally replace a tyrosine in such a targeting signal. It is possible that the YXXL/I sequences in the ITAM of Ig-α and Ig-β also act as targeting signals and that the studied Y → F mutations of the two tyrosines of ITAM (Fig. 3F) are not sufficient to abolish this targeting function. For the FcγRIII receptor, the ITAM in the γ chain has been shown to function as a targeting signal (15).

Our data apparently contradict previous studies of a chimeric IgM-MutB-γ2bm molecule that is not targeted to the endosomal compartment (2). However, in contrast to scAgR-γ2am, the MutB-γ2bm molecule is only very inefficiently internalized and this may prevent the γ2bm tail from exerting its targeting function. The tailless mIgG2atl has the same cytoplasmic sequence (KVK) as mIgM. Both molecules are expressed on the cell surface in association with the Ig-α,Ig-β heterodimer. It is, however, possible that because of differences in the transmembrane sequence, the mIgG2a is less tightly associated with the heterodimer than the mIgM or mIgD molecules. If, during endosomal transport (perhaps promoted by endosomal pH changes), the Ig-α,Ig-β heterodimer dissociates from mIgG2a, the latter molecule would require its own cytoplasmic targeting signal to reach the endosomal compartment. The transport of a tailless mIgG2atl molecule would thus not occur. The above scheme could also explain why a mIgM molecule that carries a Y → F mutation in its transmembrane part and presumably is less tightly associated with the Ig-α,Ig-β heterodimer than wild-type mIgM does not reach the antigen-presentation compartment (16).

B cells require T cell help for the production and affinity maturation of antibodies against protein antigens (17). Our finding that B cells with a tailless IgG2a-BCR cannot efficiently present antigen to T cells explains the reduction in IgG1 and IgE serum concentrations in mutant mice that express tailless IgG1-BCR and IgE-BCR, respectively (18). Activated memory B cells appear to require the cytoplasmic tails of mIg molecules and the efficient antigen presentation connected with this structure in order to expand and differentiate into antibody-producing plasma cells. Thus, the conserved COOH-terminal sequences of mIg molecules are finally assigned an important immunological function.

  • * These authors contributed equally to this work.

  • Present address: Institut Curie, 12 rue Lhomond, 75005 Paris, France.

  • To whom correspondence should be addressed. E-mail: reth{at}immunbio.mpg.de

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