Fate of the mutated IgG2 heavy chain: lack of expression of mutated membrane-bound IgG2 on the B cell surface in selective IgG2 deficiency

Tomoyoshi Terada, Hideo Kaneko, Toshiyuki Fukao, Hideaki Tashita, Ai Lian Li, Masao Takemura1 and Naomi Kondo

Department of Paediatrics and
1 Clinical Laboratory, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan

Correspondence to: Correspondence to: T. Terada


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IgG2 deficiency is clinically characterized by sinopulmonary infections caused by Pneumococcus and Hemophilus. We reported homozygous one-base insertion (1793insG) in the C{gamma}2 gene in two Japanese siblings in whom serum IgG2 levels were under detection limits. The 1793insG was present in exon 4, just upstream from the alternative splice site for M exons; the result being a complete amino acid change in transmembrane and cytosolic parts of membrane-bound {gamma}2 heavy chain (m {gamma} 2HC). To determine why this mutation caused selective and complete IgG2 deficiency, we constructed expression vectors of normal and mutant membrane-bound chimeric IgG heavy chain cDNAs. Stable transformants, Ag8N-L and Ag8M-L, expressing either normal and mutant chimeric IgG heavy chain with light chain respectively were obtained using P3X63Ag8653 as recipient cells. Of the Ag8N-L, 22.1% were surface IgG+; however, none of the Ag8M-L were surface IgG+. Addition of an anti-human IgG antibody induced cell death of Ag8N-L and we considered that the expressed chimeric IgG protein on Ag8N-L might function as the Ig receptor for signal transduction. However, Ag8M-L did not express mutant IgG on its surface nor did it secrete this mutant into culture medium. The mutant chimeric IgG protein was rapidly degraded within Ag8M-L. Thus, the mutated IgG2 heavy chain in our patient could not be expressed on the cell surface because of loss of the transmembrane domain and the evolutionally conserved cytoplasmic domain. In humans, B cells expressing surface IgG are indispensable for secretion of IgG.

Keywords: IgG2 deficiency, membrane-bound Ig, signal transduction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B lymphocytes arise from pluripotent stem cells and progress through a series of differentiation stages before final maturation into antibody-secreting plasma cells (1,2). Ig genes are constructed through Ig heavy chain and light chain rearrangements, and are expressed as membrane-bound or secreted types throughout various stages of differentiation of B cells.

B cells stimulated with antigen often undergo isotype switching leading to expression of heavy chains of other classes. From findings in knockout mice, membrane-bound Ig expression has a crucial role in the generation of efficient primary and secondary Ig responses. The primary Ig responses as well as the expansion, maintenance, or both, of Ig-bearing memory B cell depends largely on the cytoplasmic tail of the heavy chain in mice (35).

The B cell antigen receptor (BCR) is formed by the non-covalent association of membrane Ig with a heterodimer consisting of covalently linked Ig{alpha} and Igß chains. Ig{alpha} and Igß are essential for expression of membrane Ig on the cell surface. When B cells are stimulated with specific antigen, various signals are transduced via the BCR into the B cells (69). Cross-linking of surface Ig on B cells induces growth arrest and apoptosis. However, if there is concomitant ligation of CD40, B cells are rescued from anti-Ig-induced cell death (1013).

We reported a homozygous one-base insertion (1793insG) in the C{gamma}2 genes in two Japanese siblings in whom serum IgG2 levels were under detection limits. The 1793insG was present in exon 4, just upstream from the alternative splice site for M exons; the result being a complete amino acid change in transmembrane and cytosolic parts of m {gamma} 2HC (14). Why this mutation causes selective and complete IgG2 deficiency remains to be determined.

To better understand the function of mutated m {gamma} 2HC, we established stable transformants, designated Ag8N-L and Ag8M-L, expressing either normal or mutant chimera IgG heavy chain respectively, co-transfected with light chain, and the expression of normal and mutated m {gamma} 2HC and signal transduction were investigated. We also studied mechanisms of degradation of m {gamma} 2HC and signal transduction of Ag8N-L.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
cDNA amplification of the membrane bound form of the C{gamma}2 heavy chain
The normal cDNA fragment for the membrane-bound form of C{gamma}2 heavy chain was obtained as described (14). The fragment was cloned into pT7BlueT vector (Novagen, Madison, WI).

Construction of the chimeric IgG heavy chain cDNAs
A pUC19 plasmid, containing human sperm IgG1 heavy chain cDNA (secreted form), was kindly donated by Dr H. Sawai (Hyogo Medical College, Nishinomiya, Japan) (15,16). The cDNA was cloned into an expression vector, BCMGSHyg vector, and designated as BCMGSHyg-IgG1 heavy chain. There is a high sequence homology between the above cDNA for the IgG1 secreted form and that for the IgG2 membrane-bound form, and they share a SmaI site in exon 4. Hence, we obtained a chimeric normal cDNA; the sequences upstream and downstream from the SmaI site were those of IgG1 and IgG2 respectively. A chimeric mutant cDNA with 1793insG was prepared on the normal cDNA in pUC19 by side-directed mutagenesis Fig. 1Go). Normal and mutant chimeric cDNAs were cloned into BCMGSHyg vectors, and designated as BCMGSHyg-normal heavy chain and BCMGSHyg-mutant heavy chain respectively. Expression vectors for light chain cDNA in a BCMGSNeo vector (BCMGSNeo-LC) were obtained from Dr H. Sawai. The BCMGSNeo and BCMGSHyg vectors carry the neomycin- and hygromycin-resistance genes respectively.



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Fig, 1. Restriction map of heavy chain and light chain cDNA. (1) IgG1 heavy chain cDNA is 1.6 kb in length and contains a 1419 bp open reading frame. (2) Light chain cDNA is 0.9 kb in length and contains a 699 kb open reading frame. (3) Normal membrane-bound {gamma}2 heavy chain cDNA is 0.9 kb in length. (4) Chimeric normal IgG heavy chain cDNA is 1.8 kb in length. (5) Chimeric mutant IgG heavy chain mutant cDNA is 1.8 kb in lengpppth. L, leader sequence; V, variable region; D, diversity segment; J, joining segment; C, constant region; and M, membrane exon.

 
Cell lines
A mouse myeloma cell line, P3X63Ag8653 (17), which does not produce Ig, was grown in RPMI 1640 medium (Sankyo Junyaku, Tokyo, Japan) supplemented with 10% FCS, 100 µg/ml streptomycin sulfate, 20 U/ml penicillin G potassium, 2 mM L(+)-glutamine (Wako Junyaku, Osaka, Japan) and 10 mM HEPES (Dojindo, Kumamoto, Japan) (RPMI/FCS).

DNA transfection and stable transformants
Transfection was performed using Lipofectin (Gibco/BRL, Gaithersburg, MD), according to the manufacturer's instruction. Then, 2 µg each of BCMGSHyg-normal or -mutant heavy chain and BCMGSNeo light chain were co-transfected into P3X63Ag8653 cells at a concentration of 1x106 cells/ml, and designated as Ag8N-L and Ag8M-L. G418 and hygromycin selection was started 48 h after transfection. Stable transformants were maintained in the same medium with G418 (geneticin) (0.4 mg/ml of medium; Sigma, St Louis, MO) and hygromycin B (0.7 mg/ml of medium; Sigma). Finally, 2 µg each of BCMGSHyg-IgG1 heavy chain and BCMGSNeo light chain were co-transfected into P3X63Ag8653 cells, and designated as Ag8G1-L.

Semiquantitative RT-PCR for chimeric IgG heavy chain mRNAS
Total cellular RNA was extracted from 5.0x106 cells using Isogen (Nippon, Gene, Tokyo, Japan). Total RNA (5 µg) was used for cDNA synthesis. PCR amplification was carried out with primers (the forward primer was in exon 4 of {gamma}2 and the reverse primer was in M2 exon of {gamma}2) and cycling conditions as follows: forward, 5'-CGGCTCCTTCTTCCTCTACA-3', reverse, 5'-CATGTTCCTGTAGTCGGGGACGATGGTCTG-3'; 94°C for 1 min, 57°C for 1 min and 72°C for 1 min with 24, 28, 32 and 35 cycles; ß-actin-forward, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', ß-actin-reverse, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3', 94°C for 1 min, 54°C for 1 min and 72°C for 1 min with 16, 20, 24 and 28 cycles. Products were electrophoresed on a 1% agarose gel.

Characterization of surface IgG protein on Ag8N-L
MACS using mouse anti-human IgG microbeads (Militenyi Biotec, Bergisch Gladbach, Germany) was performed to concentrate surface IgG+ cells from Ag8N-L (Ag8N-Lposi). Ag8N-Lposi cells were cultured with various concentrations (final concentrations were 0, 0.1, 1 and 10 µg/ml) of anti-human IgG ({gamma} chain specific antibody (Kirkegaard & Perry, Gaithersburg, MD) or irrelevant antibody (anti-human IgA, {alpha} chain specific) (Jackson, West Grove, PA) and cultured for 3 days. Then, cells were incubated with polyclonal goat anti-human IgG antibody FITC-conjugated at 4°C and flow cytometry analysis was performed as described.

Next, Ag8N-Lposi cells were cultured as described above and harvested, and 0.2% Trypan blue and RPMI 1640 medium (1:9 v/v) were added.

In addition, Ag8N-Lposi cells were cultured as described above and [3H]-thymidine (Amersham, Little Chalfont, UK) was added to the culture medium followed by incubation for 4 h, after which the cells were harvested and [3H]thymidine incoporated by the cells was measured using a ß counter.

Flow cytometry analysis
Transformants were incubated with FITC-conjugated polyclonal goat anti-human IgG antibody (MBL, Nagoya, Japan) at 4°C for 30 min, and then washed with 1xPBS several times and analyzed using a FACScan (Becton Dickinson, Mountain View, CA). Lysis II software was used for analysis. Viable cells were gated by forward and side scatter.

Immunoblot analysis
Transformants (1x107 cells) were washed with 1xPBS, resuspended in 200 µl of extraction buffer (50 mM NaPB, pH 8.0, 0.1% Triton X, 200 mM NaCl) and sonicated. The preparations were centrifuged and separated into supernatant and pellets. Proteins were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Amersham). Immunoreactive proteins were detected using a 1:5000 dilution of goat peroxidase-conjugated anti-human IgG antibody (Jackson) and an ECL detection kit. Blots were exposed to film for 1 min.

ELISA
The double-antibody sandwich ELISA was used to detect chimera IgG heavy chains in culture supernatants of the transfectants. The wells of a 96-well plate were coated with goat anti-human IgG ({gamma} chain specific) antibody (Kirkegaard & Perry). Culture supernatants were added to each well. The culture supernatants of the Ag8G1-L served as a positive control and the supernatant of P3X63Ag8653 cells as a negative control. Secreted Ig was detected by peroxidase-labeled goat anti-human IgG ({gamma} chain specific) antibody (Kirkegaard & Perry).

Pulse–chase analysis
For pulse–chase labeling experiments, P3X63Ag8653 and transformants were pre-incubated in the RPMI/FCS medium without methionine. The cells were labeled for 1 h with 100 µCi/ml [35S]methionine (ICN, Costa Mesa, CA), and then chased for 0, 6, 24 and 72 h. At each time point, cells were collected, washed twice in PBS and lysed in 10 mM Tris, pH 7.5, 2 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 10 mM methionine with 1% BSA solution and protease inhibitors (8 µg/ml of leupeptin and 8 µg/ml of aprotinin). IgG were immunoprecipitated with Protein A (Sigma) and Protein G agarose (Boehringer Mannheim, Mannheim, Germany). Immunoprecipitates were separated on a 10% SDS–PAGE gel and subjected to autoradiography.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Construction of cDNA for the membrane-bound form of the chimeric IgG heavy chain
To investigate molecular events using the mutant m {gamma} 2HC, expression analysis has to be performed. Since Sawai et al. showed the expression of a secreted form of human IgG1 heavy chain, we used this same cDNA to construct a chimeric cDNA (15,16). The 5' area of the chimeric cDNA was a VDJ region and a constant exon 1 region corresponding to a SmaI site in exon 3 from the IgG1 heavy chain construct and the 3' area of it was from normal m {gamma} 2HC. This construct was expected to produce a complete membrane-bound chimeric IgG heavy chain. A chimeric mutant cDNA with 1793insG was prepared on normal cDNA in pUC19 using site-directed mutagenesis. These normal and mutant chimeric cDNAs were then cloned into BCMGSHyg vectors respectively (BCMGSHyg-normal and -mutant heavy chain).

Next, BCMGSHyg-normal and -mutant heavy chain constructs with the BCMGSNeo light chain construct were introduced into P3X63Ag8653 cells, using Lipofectin, according to the manufacturer's instructions (Ag8N-L).

Semiquantitative determination of Ag8N-L and Ag8M-L mRNA using RT-PCR
Semiquantitative PCR was performed to examine expression of normal and mutant heavy chain in Ag8N-L and Ag8mM-L. The level of expression of the mutant heavy chain of mRNA was almost the same as that of the normal mRNA (Fig. 2Go).



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Fig. 2. Semiquanitative determination of normal and mutant heavy chain in Ag8N-L and Ag8M-L mRNA using RT-PCR. Total RNA (5 µg) was used for cDNA synthesis followed by PCR using primers described in Methods. In each case, 24, 28, 32 and 35 cycles were run in Ag8N-L and Ag8M-L. ß-Actin was used as a control with a run of 16, 20, 24 and 28 cycles. The level of expression of the mutant heavy chain of mRNA was almost the same as that of the normal mRNA.

 
Normal chimeric IgG heavy chain was functionally expressed on the surface of P3X63Ag8653
Flow cytometry analysis showed that 22.1% of the Ag8N-L were surface IgG+; however, none of the Ag8M-L was surface IgG+ (Fig. 3Go). To determine if the Ig receptor on Ag8N-L was functional, surface IgG+ Ag8N-L was concentrated using magnetic beads (Ag8N-Lposi). About 80% of Ag8N-Lposi were surface IgG+. The cells were incubated with various concentrations (final concentrations were 0, 0.1, 1 and 10 µg/ml) of anti-human IgG antibody to Ag8N-Lposi cells and cultured for 3 days. The cross-linked Ig receptor reduced the number of surface Ig+ cells (Fig. 4AGo). When Ag8N-Lposi cells were incubated without antibody and cultured for 3 days, viability of Ag8N-Lposi cells was about 60%. This result showed that some Ag8N-Lposi cells were dying spontaneously. Thus, Ag8N-Lposi cells were incubated with various concentrations (final concentrations were 0, 0.1, 1 and 10 µg/ml) of irrelevant antibody and cultured for 3 days. In spite of the addition of irrelevant antibody, viability of Ag8N-Lposi cells was not changed. The reduced viability and thymidine incorporation of Ag8N-Lposi cells stimulated by anti-human IgG antibody were caused by cross-linking of the Ig receptor. The ratio of dead cells depended on the concentration of Ig antibody (Fig. 4B and CGo). These results indicate that the Ig receptor expressed on the myeloma cell line is functional and that cross-linking of the Ig receptor on the myeloma cell line led to cell death.



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Fig. 3. Flow cytometry analysis of IgG expression on Ag8N-L and Ag8M-L. (A) Ag8N-L without antibody. (B) Ag8N-L stained with FITC-labeled anti-human IgG antibody. (C) Ag8M-L without antibody. (D) Ag8M-L stained with FITC-labeled anti-human IgG antibody. Of the Ag8N-L, 22.1% were surface IgG+ (B). On the other hand, none of the Ag8M-L were surface IgG+ (D).

 


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Fig. 4. Ag8N-Lposi cells were stimulated by various concentrations of anti-human IgG antibody (0, 0.1, 1 and 10 µg/ml) for 3 days and analyzed by flow cytometry. Surface IgG+ cells were 50.3, 46.7, 32 and 16% respectively. (B) Ag8N-Lposi cells were stimulated by various concentrations of anti-human IgG antibody (0, 0.1, 1 and 10 µg/ml) or irrelevant antibody (0, 0.1, 1 and 10 µg/ml) for 3 days and stained with 0.2% Trypan blue. Ag8M-L was also stimulated as described above. Viability of Ag8N-Lposi cells stimulated by anti-human IgG antibody was reduced and the ratio of dead cells depended on the concentration of Ig antibody. Viability of Ag8N-Lposi cells stimulated by irrelevant antibody and Ag8M-L cells stimulated by anti-human IgG antibody or irrelevant antibody was not changed by the concentration of antibody. •, Ag8N-Lposi cells stimulated by anti-human IgG antibody (0, 0.1, 1 and 10 µg/ml); ö, Ag8N-Lposi cells stimulated by irrelevant antibody (0, 0.1, 1 and 10 µg/ml; {blacksquare}, Ag8M-L cells stimulated by anti-human IgG antbody (0, 0.1, 1 and 10 µg/ml). {square}, Ag8M-L cells stimulated by irrelevant antibody (0, 0.1, 1 and 10 µg/ml). (C) Ag8N-Lposi cells were stimulated by various concentration of anti-human IgG antibody (0, 0.1, 1, 10 µg/ml) or irrelevant antibody (0, 0.1, 1, and 10 µg/ml) for 3 days and the thymidine incorporation assay performed. [3H]Thymidine incorporated by Ag8N-Lposi cells stimulated by anti-human IgG antibody was reduced when the concentration of IgG antibody was increased. [3H]Thymidine incorporated by Ag8N-Lposi cells stimulated by irrelevant antibody and Ag8M-L cells stimulated by anti-human IgG antibody or irrelevant antibody was not changed by the concentration of antibody. •, Ag8N-Lposi cells stimulated by anti-human IgG antibody (0, 0.1, 1 and 10 µg/ml); ö, Ag8N-Lposi cells stimulated by irrelevant antibody (0, 0.1, 1 and 10 µg/ml); {blacksquare}, Ag8M-L cells stimulated by anti-human IgG antibody (0, 0.1, 1 and 10 µg/ml); {square}, Ag8M-L cells stimulated by irrelevant antibody (0, 0.1, 1 and 10 µg/ml).

 
Chimeric mutant IgG heavy chain was degraded in the Ag8M-L cells
Western blot analysis showed that heavy and light chains of Ag8N-L were present in the homogenate supernatant and pellet fraction (Fig. 5Go, lanes 2 and 5), but the heavy chain of Ag8M-L was not evident (Fig. 5Go, lanes 3 and 6). Heavy and light chains of Ag8N-L and Ag8M-L were not evident in the culture supernatant.



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Fig. 5. Immunoblot analysis of chimeric IgG protein in Ag8N-L and Ag8M-L cells. Cell lysis (30 µg/lane) and pellets after sonication were separated on 10% SDS–PAGE gel and analyzed by immunoblotting to detect normal and mutant heavy chains. Lane 1, homogenate supernatant of P3X63Ag8653 cells; lane 2, homogenate supernatant of Ag8N-L cells; lane 3, homogenate supernatant of Ag8M-L cells; lane 4, pellet fraction of P3X63Ag8653 cells; lane 5, pellet fraction of Ag8N-L cells; lane 6, pellet fraction of Ag8M-L cells. Western blot analysis showed that heavy and light chains of Ag8N-L were present in the homogenate supernatant and pellet fraction (lane 2 and 5), although the heavy chain of Ag8M-L was not evident (lanes 3 and 6).

 
ELISA showed no evidence of the mutant IgG heavy chain in the culture supernatant, although secreted IgG1 was present in the supernatant of Ag8G1-L (data not shown). Pulse–chain analysis showed that the normal heavy chain of Ag8N-L could be detected for up to 6 h but mutant heavy chain of Ag8M-L was faint (Fig. 6Go). As the mutant IgG heavy chain was not detected in Ag8M-L cells, culture medium or on the cell surface, we considered that the mutant IgG heavy chain degraded rapidly in the cytoplasm of Ag8M-L cells.



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Fig. 6. Pulse–chase analysis of chimeric IgG protein in Ag8N-L and Ag8M-L cells. For pulse–chase labeling experiments, P3X63Ag8653 and transformants were preincubated in RPMI/FCS without methionine. The cells were labeled for 1 h with 100 µCi/ml[35S]-methionine, and then chased for 0, 6, 24 and 72 h. Lane 1, P3X63Ag8653 (1 h pulse); lane 2, P3X63Ag8653 (6 h chase); lane 3, Ag8G1-L (1 h pulse); lane 4, Ag8G1-L (6 h chase); lane 5, Ag8M-L (1 h pulse); lane 6, Ag8M-L (6 h chase); lane 7, Ag8N-L (1 h pulse); lane 8, Ag8N-L (6 h chase). Pulse–chase analysis showed that the normal heavy chain of Ag8N-L could be detected for up to 6 h (lane 5 and 6) but the mutant heavy chain of Ag8M-L was faint (lanes 7 and 8). IgG was not evident at 24 or 72 h. Arrowhead indicates the mutant heavy chain which is slightly larger than the wild heavy chain.

 
In addition, Ag8M-L was maintained at 30°C to determine if the mutant chimeric IgG protein was degraded. Results of flow cytometry analysis, immunoblots, ELISA and pulse–chase analysis were much the same when Ag8M-L was maintained at 37°C (data not shown). These results suggest that mutant IgG protein cannot be rescued when activation of some proteases is disturbed at 30°C.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We earlier identified a gene mutation, a homozygous one-base insertion (1793insG), in the C{gamma}2 gene, in patients with IgG2 deficiency (14). It remained to be explained why this mutation causes selective and complete IgG2 deficiency. At least two possibilities could explain the mechanism of IgG2 deficiency with the frame shift of m {gamma} 2HC. One is that this mutation results in complete loss of function and structure as a BCR, and the mutant m {gamma} 2HC never appears on the B cell surface. The other is that the mutant m {gamma} 2HC which has lost the conserved motif in the cytoplasmic tail could be expressed on the B cell surface but could not complete signal transduction or antigen processing. Hydropathy profiles of normal and mutant m {gamma} 2HC sequences encoded by M exon were investigated, according to Kyte and Doolittle (18) (Fig. 7Go). The average hydropathy of 19-residue segments of the normal m {gamma} 2HC transmembrane domain was >+2, satisfying a condition for membrane-spanning sequences. On the other hand, most of the mutant sequence encoded by M exons was hydrophobic and the average hydropathy of any 19-residue segment of the mutant sequence was <+1.2, which suggests that there is no membrane-spanning sequence in the mutant sequence (14).



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Fig. 7. Hydropathy profile of normal and mutant m {gamma} 2HC. The average hydropathy of 19-residue segments of the normal m {gamma} 2HC transmembrane domain was >+2, satisfying a condition for membrane-spanning sequences. On the other hand, most of the mutant sequence encoded by M exons was hydrophobic and the average hydropathy of any 19-residue segment of the mutant sequence was <+1.2.

 
For clarification, we established Ag8N-L and Ag8M-L to analyze character and function of the mutated m {gamma} 2HC. Flow cytometry analysis revealed that the mutant IgG heavy chain could not be expressed on the cell surface. Next, we investigated localization of mutant IgG2 in myeloma cells. This heavy chain was not evident either in the cell or in the supernatant. We then analyzed newly synthesized IgG using pulse–chase analysis and found that the normal IgG heavy chain of Ag8N-L was present for up to 6 h; however, the mutant IgG heavy chain of Ag8M-L was faint for up to 6 h. Thus mutant degradation of the IgG heavy chain probably occurred in the cell.

If the patient's B cells undergo isotype switching to IgG2 and express m {gamma} 2HC, mutant m {gamma} 2HC protein degradation would occur and hence would not be expressed on the cell surface, and the cell could not respond to various signals from the exterior; lack of a signal via BCR would prevent B cell proliferation and cell death would ensue. We find that expression of m {gamma} 2HC on the B cell surface is indispensable for responses to antigen and secretion of IgG2 in humans.

Cross-linking of the Ig receptor induced cell death in myeloma cells. Differentiation from B cells to myeloma cells or plasmacytes led to the down-regulation of membrane Ig. The signal from the Ig receptor expressed on myeloma cells induced cell death. Despite the stable transfectant, only 22.1% of Ag8N-L expressed IgG on the surface. Using magnetic beads, concentrated IgG+ cells (80% positive) also lost surface IgG (30%) after 3 days in culture. Surface IgG on the myeloma cells may possibly receive the signal from cross-reacting protein in serum. When we used serum-free medium for culture, the cells did not grow. Surface IgG+ myeloma cells grew more slowly than did surface IgG- myeloma cells.

There are at least two possibilities that could explain the mechanism of degradation with mutant m{gamma}2HC. Many post-translational events often require quality control for correct assembly in the endoplasmic reticulum (ER). ER quality control of malfolded membrane-associated proteins (1922), malfolded soluble secretory proteins (2325) and non-mutant secretory proteins (2628), but not Ig, has been demonstrated (29). Correct assembly is regulated by the activity of ER-specific chaperones and by folding enzymes. If assembly of the Ig molecule is incorrect, Ig cannot be transported from the ER. The heavy chain of IgM and {gamma} chain of HLA-DR is such a case (30,31). It was suggested that mutant m {gamma} 2HC degradation occurs by activity of ER-specific chaperones in the lumen of ER because of incorrect assembly of Ig molecules. In IgM-secreting B cells where the L chain is absent, the H chains are retained and degradation occurs in a short time (32). One possibility is that incompletely folded or misfolded Ig are retained by ER-specific chaperones and degraded by proteases.

Mutant m {gamma} 2HC has retention/retrieval signals in the ER called the RXR motif (Fig. 8Go). This motif locates in the cytoplasmic domain of mutant m {gamma} 2HC and normal m {gamma} 2HC do not have this motif. Otherwise, in light chain-producing cells, some of the mutant heavy chains accumulate together with light chains in ER-derived vesicles and some are secreted as IgG (33). Crystallographic analyses and in vitro folding studies indicate that each chain consists of a series of Ig domains that fold independently of each other (34,35). The {alpha} and ß subunits of the ATP-sensitive K+ channel (KATP) have the RXR motif. However, when subunits assemble correctly, RXR motifs are masked and protein is transported to the cell surface (36). Thus, another possibility is that mutant m {gamma} 2HC can assemble completely in the ER, except for M exon, and be transported to the Golgi apparatus. However, mutant m {gamma} 2HC cannot be transported to the cell surface because it has the RXR motif in the cytoplasmic domain and degradation occurs in the Golgi apparatus or in the ER because mutant m {gamma} 2HC are retrieved by the ER.



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Fig. 8. Amino acid sequences encoded by M exon for normal and mutant m {gamma} 2HC. The one-base insertion resulted in a frame shift of m {gamma} 2HC, and generated a completely different amino acid sequence of the transmembrane and intracellular portion. The hydrophobic amino acids, cluster and conserved cytoplasmic tail residues (Lys–Val–Lys motif and Tyr–X–X—Met motif) are absent in the mutant sequence. On the other hand, mutant m {gamma} 2HC has the ER retention/retrieval motif (RXR) in the cytoplasmic region (underlined).

 
Mutant m {gamma} 2HC cannot be rescued even if activation of proteases is disturbed at 30°C and it may be that degradation of m {gamma} 2HC is not caused by known proteases. By way of summary, we showed that expression of membrane Ig is indispensable for responses to antigen and for effective secretion of Ig. The lack of expression of mutant IgG2 on the cell surface can explain the selective and complete IgG2 deficiency.


    Acknowledgments
 
We thank Dr H. Sawai for the generous gift of a pUC19 plasmid, containing human sperm IgG1 heavy chain cDNA and BCMGSNeo-light chain, and M. Ohara for helpful comments. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.


    Abbreviations
 
Ag8GI-L BCMGSHyg-IgG1 heavy chain and BCMGSNeo light chain were co-transfected into P3X63Ag8653
Ag8M-L BCMGSHyg-mutant heavy chain and BCMGSNeo light chain were co-transfected into P3X63Ag8653
Ag8N-L BCMGSHyg-normal heavy chain and BCMGSNeo light chain were co-transfected into P3X63Ag8653
Ag8N-Liposi MACS using mouse anti-human IgG microbeads was performed to concentrate surface IgG+ cells from Ag8N-L
BCR B cell receptor
ER endoplasmic reticulum
m {gamma} 2HC membrane-bound {gamma}2 heavy chain

    Notes
 
Transmitting editor: T. Watanabe

Received 27 October 2000, accepted 9 November 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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