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The Journal of Immunology, 2006, 177: 1139-1145.
Copyright © 2006 by The American Association of Immunologists

HS1-Associated Protein X-1 Interacts with Membrane-Bound IgE: Impact on Receptor-Mediated Internalization1

Iris Oberndorfer*, Doris Schmid*, Roland Geisberger*, Gertrude Achatz-Straussberger*, Reto Crameri{dagger}, Marinus Lamers{ddagger} and Gernot Achatz2,*

* Department of Molecular Biology, University of Salzburg, Salzburg, Austria; {dagger} Swiss Institute of Allergy and Asthma Research, Davos, Switzerland; and {ddagger} Max Planck Institute for Immunobiology, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
From the numerous surface markers of a B cell, the BCR is probably the most powerful one in influencing developmental processes. The extracellular Ag-binding moiety of the BCR consists of the membrane-bound Ig (mIg),3 a tetramer of a H chain homodimer and either two {kappa} or two {lambda} L chains. Two further domains, the membrane-spanning domain (coded by exon M1) and the cytoplasmic tail (coded by exon M2), provide the prerequisite for later membrane localization. All mIg isotypes are noncovalently associated with a heterodimer of two transmembrane proteins, Ig{alpha} (CD79a) and Igbeta (CD79b) (1). The Ig{alpha}/Igbeta molecules belong to the Ig superfamily and carry ITAM (consensus: YxxL/Ix6–8YxxL/I) in their cytoplasmic tails (2, 3). Receptor stimulation initiates a complex cascade of cytoplasmic-signaling events resulting in the appropriate cellular response. Interestingly, none of the cytoplasmic tails of the various Ig isotypes contain ITAM motifs or ITIMs (consensus: I/V/L/SxYxxL/V) (4). Thus, a signaling cascade committed by the cytoplasmic tails of mIgs itself seems to be unlikely, but rather postulates proteins that actively bind the cytoplasmic tails of mIgs (5, 6, 7).

The earliest detectable biochemical event that follows BCR aggregation is increased activity of protein tyrosine kinases of the Src family (Lyn), resulting in phosphorylation of the tyrosine residues within the ITAMs of Ig{alpha} and Igbeta and the non-ITAM tyrosine 204 of Ig{alpha} (8), subsequently followed by the initiation of several signaling pathways (reviewed in Refs. 6 , 9 , and 10). Syk, after activation by Lyn, tyrosine-phosphorylates the hemopoietic linage cell-specific protein 1 (HS1), which is specifically expressed in hemopoietic cells. HS1 is a 75-kDa hemopoietic linage-specific protein (11, 12), with known functions in B cell proliferation and BCR-induced apoptosis (11). After tyrosine phosphorylation, HS1 translocates from the cytoplasm to the nucleus (11). HS1 was shown to interact with HS1-associated protein X-1 (HAX-1, also known as HS1-binding protein) (13), a 35-kDa, ubiquitously expressed protein. However, the subcellular localization of HAX-1 depends on the cell type. Accordingly, HAX-1 was localized in the cytoplasm (13) and even in mitochondria (14), and was found along the endoplasmic reticulum and the nuclear envelope as well (15). HAX-1 displays a weak homology to the intracellular mammalian protein, BNip3 (13), and similarity to the functionally important domains BH1 and BH2 of Bcl-2 family member proteins. An internal proline-glutamic acid-serine-threonine sequence suggests that the HAX-1 protein may be degraded rapidly (16).

Besides its association with HS1, HAX-1 was also reported to physically associate with several other molecules indicating that the biological function of HAX-1 can roughly be divided into three categories: 1) Association of HAX-1 with HS1 (13), Kaposi’s sarcoma-associated herpes virus K15 (17), Epstein-Barr nuclear Ag 5 (EBNA5 or EBNA-LP) (18, 19), Bcl-2 and BHRF1 (EBV homolog of Bcl-2) (20), Omi/HtrA2, a nuclear-encoded mitochondrial serine protease with proapoptotic function (14) and HIV protein Vpr (21) indicates the involvement of HAX-1 in the regulation of apoptotic processes. 2) Association of HAX-1 with cortactin (or EMS1) (15), polycystic kidney disease protein 2 (15), ATP-binding cassette transporters BSEP, MDR1, and MDR2 (22) and G{alpha}-13, a cell migration-stimulating component (23) emphasizes the involvement of HAX-1 in cell motility processes.

3. Finally, physical interaction of HAX-1 with the 3' untranslated region of human vimentin mRNA (24) indicates RNA-binding capacity and, as interaction factor of the IL-1{alpha} N terminus (25), HAX-1 seems to act as cytoplasmic retention factor.

In summary, HAX-1 performs a multifunctional impact on biological processes. In the current work, we present HAX-1 as novel interaction partner of the cytoplasmic tail of mIgE. We show evidence that the cytoplasmic tail of IgE serves as docking site for interacting proteins thereby affecting Ag processing and presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

Monoclonal mouse-IgG1 anti-HAX-1 Ab was purchased from BD Transduction Laboratories (BD Biosciences), monoclonal mouse-IgG1 anti-penta-His Ab from Qiagen, monoclonal mouse IgG1 anti-{alpha}-tubulin Ab from Abcam, and bovine anti-mouse IgG1-conjugated with HRP as well as rat-anti-CD19-RPE was purchased from Serotec. Polyclonal anti-murine IgE serum was raised in rabbits against purified mouse IgE, {kappa} (BD Pharmingen) and goat-anti-rabbit-IgG (whole molecule)-alkaline phosphatase conjugate was purchased from Sigma-Aldrich. Rat (IgG1)-anti-murine IgE clone 51.3 (anti-CH4-domain of IgE) was provided by Dr. T. Waks (Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel) (26).

Cell lines and media

J558L-mb1 (gift from Dr. J. Wienands, Department of Biochemistry, University of Bielefeld, Germany.) and JW813/4 BCR– were grown in RPMI 1640 medium supplemented with 10% FCS, 55 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. JW813/4 BCR+ medium was additionally supplemented with 25 mg/L mycophenolic acid, 250 mg/L xanthine, and 13.6 mg/L hypoxanthine.

Cell line JW813/4 BCR+ small-interfering RNA (siRNA) was stably transfected with pU6-siRNA plasmids (Biomyx Technology) and selected with 400 ng/ml geneticin (G418 sulfate).

Production and purification of recombinant proteins

DHFR, CH4, DHFR tail, CH4 tail. The cytoplasmic tail of mIgE (YGATVTVLKVKWVFSTPMQDTPQTFQDYANILQTRA) was C-terminally fused to the constant region H chain (CH)4 domain of mouse or mouse dihydrofolate reductase (DHFR), respectively. CH4 and DHFR functioned as carrier proteins for the tail peptide. The cDNA sequences were cloned into pHIS-parallel vectors (27) leading to N-terminally His-tagged proteins. Recombinant His-tagged proteins, designated as CH4, DHFR, CH4 tail, and DHFR tail, were batch-purified under denaturing conditions in the presence of 8 M urea using nickel-nitrilotriacetic acid agarose.

Murine HAX-1. Murine HAX-1 cDNA-sequence was amplified from a murine cDNA-library by PCR using primers: no. 288 forward (fwd), 5'-ATGAGCGTCTTTGATCTTTTCCGAGGC and no 289 reverse (rev), 5'-CTATCGGGACCGAAACCAACGTCCTAG.

The PCR product was ligated into the pPCR-Script AMP SK+ plasmid (Stratagene) according to the manufacturer’s protocol. After sequence confirmation HAX-1 was recloned into the pHIS-parallel 2 vector. After nickel-nitrilotriacetic acid purification, the protein was dialyzed against 10 mM NaH2PO4 (pH 7.5).

Construction of a murine B cell cDNA library in a pJuFo-phagemid vector

Female BALB/c mice were repeatedly immunized with 5 µg of recombinant Bet v 1a in 3–4 wk intervals. Lymph nodes (axial and inguinal) and the spleen were prepared from two mice erythrocyte lysis was performed. The splenocytes and the monodisperse lymph node cell preparation were pooled and cultured at a density of 106 cells/ml in RPMI 1640 complete medium supplemented with 15 µg/ml LPS, 10 µg/ml recombinant Bet v 1a, and 500 U/ml recombinant mouse IL4 (BD Pharmingen). After 3 days, lymphocytes were stained with anti-CD19-PE Ab and CD-19+ B cells were sorted with MACS technique yielding ~4 x 107 CD-19+ B cells of >95% purity. mRNA was isolated with MACS oligo(dT) Micro beads. Isolated mRNA was transcribed into cDNA using the Stratagene cDNA synthesis kit. The cDNA was ligated (EcoRI/XhoI) into a modified pJuFo vector (28), designated as pGA110 (5). The library contained ~5 x 105 single transformants with insert size ranging between 1400 and 2400 bp. Inserts were sequenced using the following primers: no. 220 fwd, 5'-GCAAACCGAAATCGCGAACCTGC and no. 214 rev, 5'-GTAAAACGACGGCCAGTG.

Phage display biopanning

Phage was generated as described previously (28, 29, 7). The cytoplasmic tail was fused to the carrier proteins DHFR or CH4 domain of IgE, respectively. Carrier-specific phage was depleted by preincubation of the phage preparation with an excessive amount of the respective carrier protein before each panning round. Four different biopanning experiments with different bait proteins were performed. Decreasing amounts of recombinant tail fusion proteins (200, 150, 100, and 100 µg in the first, second, third, and fourth panning round, respectively) were immobilized on MaxiSorp Immuno tubes. Immobilization was done in denaturing buffer (8 M urea, 50 mM NaH2PO4 (pH 8), 300 mM NaCl) over night at 4°C. Proteins were refolded "in situ" by washing the immunotubes once with 5 M urea in PBS (pH 7.5) followed by washing twice with PBS. For the phage selection, preincubated phage was transferred into the blocked tubes. Eluted phage was used for infection of TG1 Escherichia coli cells for a successive panning round. Specific phage were detected with a "Phage" ELISA by adding serially diluted phage (3.511 to 1.110 PFU/ml) to the CH4 tail or CH4-coated wells and subsequent staining with anti-M13-HRP Ab (Amersham Pharmacia Biotech). OD values measured with CH4 as Ag were subtracted from signals measured with the CH4 tail to obtain tail-specific signals.

Real-time PCR

Measurement of HAX-1 enrichment in phage display. The relative amounts of HAX-1-encoding phagemids obtained after each panning round were determined by real-time PCR using a HAX-1-specific primer-set: no. 292 fwd, 5'-TAGACAGTGAGGGCCGGAGGGAGAC; no. 293 rev, 5'-TGGCAATGGGCAACAGGAAGGGAGTGG; and a pGA110 vector-specific primer set: no. 290 fwd, 5'-CGGCGGCTCTGGTGGTGGTTCT; no. 291 rev, 5'-ACTGTAGCGCGTTTTCATCGGCATTTTCGGTCAT.

Phagemid preparations obtained from bacteria infected with phage from each panning round were used as templates for PCR amplification. To prove correct amplification, PCR products were sequenced. Values obtained with the vector-specific primer set were set to 100% and the percentage of HAX-1-encoding plasmids was calculated for each sample. All individual PCRs were done in duplicates and SDs were calculated from three independently performed experiments.

Measurement of HAX-1 transcripts in siRNA-transfected cells lines. The relative amounts of HAX-1 mRNA in cells transfected with siRNA targets for HAX-1 were determined using primer set (nos. 292, 293) for HAX-1 and an Arbp (60S acidic ribosomal protein P0)-specific primer set: fwd, 5'-TGCACTCTCGCTTTCTGGAGGGTG; rev, 5'-AATGCAGATGGATCAGCCAGGAAGG.

mRNA was isolated from 107 cells, cDNA was prepared from 3 µg of mRNA and finally used as template in the real-time PCR amplifications.

Coimmunoprecipitation

"In vitro" coimmunoprecipitation. A total of 2 x 107 J558L cells were resuspended in 1 ml of ice-cold lysis buffer (25 mM Tris (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM sodium orthovanadate) containing a protease inhibitor mix (Complete Mini; Roche). A total of 30 µg of carrier-tail-fusion protein (CH4 tail) or carrier protein (CH4), respectively, were added and cells were then lysed by rotating at 4°C for 45 min. Insoluble material was removed by centrifugation at 15,000 x g for 10 min at 4°C. A total of 2 µg of anti-5xHis Ab were added to the cleared lysate and left rotating at 4°C for 2 h. For precipitation 35 µl of protein G-Sepharose 4 Fast Flow (Amersham Biosciences) were added and the mixture left rotating for 2 further hours at 4°C. Sepharose was pelletized by centrifugation at 250 x g at 4°C for 5 min, and the precipitate washed twice with 1 ml of ice-cold lysis buffer and twice with 1 ml of ice-cold wash buffer (100 mM Tris (pH 8.1), 0.5 M LiCl). Immune complexes were eluted from the beads by incubation with 50 µl of 2x reducing SDS-PAGE-loading buffer (125 mM Tris, 140 mM SDS, 20% glycerol, 10% 2-ME, 30 µM bromphenol blue (pH 6.75)) for 5 min at 95°C. After removing beads by centrifugation at 15,000 x g for 3 min at 4°C, 20 µl of the cleared supernatant were loaded onto a 10%-SDS-PAGE.

"In vivo" coimmunoprecipitation. A total of 2 x 107 JW813/4 BCR+ and JW813/4 BCR– myeloma cells were lysed and the precipitation was performed as described before. Nitrophenyl (NP)-specific IgE-BCR was precipitated using 30 µl of NP-Sepharose (4-hydroxy-3-nitrophenylacetyl hapten conjugated to Sepharose via a six-carbon spacer; Biosearch Technologies) or CL-2B Sepharose (Stratagene) as a control for unspecific binding to Sepharose.

Crude cell lysates were separated by SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride (0.45 µm) membrane (standard Western protocol). For detection, primary and HRP-conjugated secondary Abs, see Reagents and Abs, were used.

Surface plasmon resonance analysis (Biacore X)

Recombinant His-tagged murine HAX-1 (60 ng/µl in 10 mM NaH2PO4 (pH 7.5)) was diluted 1/5 in 10 mM sodium acetate (pH 4) and coupled to the CM5 chip according to the manufacturer’s instructions. Approximately 4000 resonance units rHAX-1 were coupled to flow cell 2. Empty flow cell 1 served as reference. The synthesized IgE-tail peptide (1 mg/ml in PBS; sequence HHHHHHKVKWVFSTPMQDTPQTFQDYANILQTRA) was injected at different concentrations (5–100 µM) in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20 buffer (Biacore), and the surface plasmon resonance was recorded. Data were analyzed with BIAevaluation software (Biacore).

RNA interference

Two siRNA target sequences were selected via <rnaidesigner.invitrogen.com> (target 1) and <www.ambion.com/techlib> (target 8). The control target sequence was taken from Ortiz et al. (22). Selected target sequences were tested for exact matches of >12 nucleotides. Target sequences that matched mRNA sequences different from the murine HAX-1 sequence were excluded. The siRNA target sequence was cloned in forward and reverse complement orientation spaced by a loop with the sequence TTCAAGAGA. The expression of this construct leads to a RNA stem loop conformation, which contains the desired RNA double strand needed for RNA interference. For cloning purposes, restriction sites at the 5' and 3' ends were added.

HAX-1 siRNA target 1: nos. 433 and 434. No. 433, 5'-TTTGGCTACTAGGACGTTGGTTTCGTTCAAGAGACGAAACCAACGTCCTAGTAGCTTTTT; no. 434, 5'-CTAGAAAAAGCTACTAGGACGTTGGTTTCGTCTCTTGAACGAAACCAACGTCCTAGTAGC.

HAX-1-siRNA target 8: nos. 435 and 436. No. 435, 5'-TTTGGCTTAAGTACCCAGATAGTTTCAAGAGAACTATCTGGGTACTTAAGCTTTTT; no. 436, 5'-CTAGAAAAAGCTTAAGTACCCAGATAGTTCTCTTGAAACTATCTGGGTACTTAAGC.

Control target: nos. 347 and 438. No. 347, 5'-TTTGGTGTACAGCGATGTTGTCGTTCAAGAGACGACAACATCGCTGTACACTTTTT; no. 438, 5'-CTAGAAAAAGTGTACAGCGATGTTGTCGTCTCTTGAACGACAACATCGCTGTACAC.

Oligonucleotides were annealed and ligated into the double-digested (XbaI/BbsI) pU6-siRNA expression vector (Biomyx Technology). JW813/4 BCR+ myeloma cells were transfected and single transfectants were isolated via limiting dilution in selective medium with geneticin. Stable integration was tested by genomic PCR with a primer set (nos. 446 and 447) spanning the sequence from the U6 promoter to the siRNA-target sequence. No. 446 fwd, 5'-GAAGCATTTATCAGGGTTATTGTCT; no. 447 rev, 5'-TTGAGCGTCGATTTTTGTGATG.

All stable cell lines were controlled for the induction of an IFN response by measuring the induction of OAS1, a classic IFN target gene, by real-time PCR (nos. OAS1f and OAS1r). OAS1f fwd, 5'-TCCCAACTCCCGGGCTCTGAG; OAS1r rev, 5'-GCGGGGTACGCCCACTGATG.

BCR-internalization assay

Ag-internalization assays were performed with slight modifications according to Aluvihare et al. (30). A total of 1.2 x 106 logarithmically growing JW813/4 BCR+ siRNA cells were washed once with PBS and subsequently incubated on ice with anti-IgE-FITC (clone EM95-3) for 45 min in a total volume of 600 µl of cold FACS buffer (PBS 3% FCS). The cells were washed twice with ice-cold FACS buffer, resuspended in 1.2 ml of RPMI 1640 complete medium (10% FCS) and equal aliquots of 0.3 ml were incubated at 37°C in a water bath in humidified atmosphere containing 7% CO2 in an open FACS tube for the time indicated (0, 10, 20, or 30 min). The cell samples were pelletized and resuspended in 500 µl of FACS buffer each. Fluorescence intensities were analyzed using flow cytometry (FACSCalibur; BD Biosciences). The cells gated for living cells (propidium iodide). The relative fluorescence intensities were calculated by equating the fluorescence (geometrical mean) at time 0 with 100%. For calculating the amount of internalized Ag, the mean fluorescence of time 0 was set to 0%. Mean values and SDs were calculated from 10 independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of HAX-1 as interaction partner of the cytoplasmic tail of mIgE

We used the phage display biopanning technique to screen a murine CD19+ cDNA library constructed in the modified pJuFo phagemid vector pGA110 (5, 28). As bait, we used a chimeric recombinant fusion protein composed of the cytoplasmic tail of mIgE fused to the C terminus of DHFR or CH4 as carrier (DHFR tail and CH4 tail). By fusing the short tail with a carrier, we avoided sterical interference with the blocking agent and in parallel were able to mimic a "dimeric IgE tail" during the panning procedure, as present under in vivo conditions. We point to this fact because we failed to isolate tail-specific binding partners in a previous approach using a yeast two-hybrid screen (in this approach, the bait protein is obligatorily expressed as monomer; our unpublished data). Thus, we hypothesized that a potential binding partner might need the tail in its dimeric configuration. Four independent phage display experiments were performed. In panning A and B, DHFR and CH4 tails were used during all four panning rounds. In panning C and D, the carrier protein was exchanged after the first two panning rounds. After the fourth panning round of each phage display setup 30 single clones were tested in ELISA for binding to the recombinant IgE tail. Subsequently, phagemids from ELISA-positive clones were prepared and sequenced. Ten of 14 sequenced clones represented the C terminus of the HAX-1 protein. Within these 10 clones eight encoded 213 C-terminal aa of HAX-1 and two clones originated from a longer cDNA clone that coded for 231 C-terminal aa (Fig. 1). To determine the increase of HAX-1-encoding phagemids during the four panning rounds we performed real-time PCR measurements (Fig. 2) using primer sets that were specific for the phagemid vector backbone or the HAX-1-cDNA insert. In the original cDNA library, we measured 0.014% HAX-1-encoding phagemids. The extent of HAX-1 enrichment varied significantly within the four phage display setups. Only 2% of all phagemids of the fourth panning round of panning A coded for HAX-1 while 62% coded for HAX-1 in panning B. In pannings C and D, we measured an intermediate value of ~33%. The variations in enrichment efficiency may be explained by the varying efficiencies of the preincubation steps to eliminate carrier-specific phage.


Figure 1
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  		FIGURE 1. Functionally important domains inside the HAX-1 open reading frame. Upper panel, HAX-1 shows similarities to the functionally important domains of antiapoptotic proteins BH1 and BH2 (BH... Bcl-2 homology domain). The PEST sequence is a sequence motif for rapidly degraded proteins. Lower panel, Two phage-clone populations encoding 213 or 231 aa of the C-terminal part of HAX-1, respectively, were enriched during phage display. The alignment with the full-length protein shows that both clones harbor BH2 and the longer clone partially includes the BH1 domain.

 

Figure 2
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  		FIGURE 2. Enrichment of HAX-1-encoding phage during phage display. Phage eluted after each panning round were used to reinfect E. coli. Phagemids were prepared from the different populations and used as template for real-time PCR. HAX-1 was detected with primers specific for HAX-1 cDNA, whereas the plasmid backbone was detected with primers specific for a vector-encoded phage envelope protein (pIII). The relative percentage of HAX-1 in each sample was calculated. The measurement of each sample was performed in duplicates, and the real-time PCR was performed independently three times. Panning round "0" corresponds to the original cDNA library.

 
HAX-1-expressing phage recognize the recombinant mIgE-tail in ELISA

Phage, displaying either the 213 aa- or the 231 aa-long C-terminally truncated HAX-1 protein (Fig. 2) were tested in ELISA for in vitro binding of the recombinant mIgE tail. As negative control, we used the Niemann Pick C2 protein. ELISA plates were coated with 100 ng of purified recombinant protein per well (CH4 or CH4 tail, respectively). Specific phage were detected with an anti-M13-HRP Ab. Absorbance values were recorded at 405 nm. OD values measured with CH4 as Ag were subtracted from signals measured with the CH4 tail to obtain tail-specific signals. Both HAX-1 clones bound specifically to the tail but phage particles displaying the longer HAX-1 fragment gave higher signals than the shorter (Fig. 3).


Figure 3
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  		FIGURE 3. Titration curves of HAX-1-expressing phage in ELISA. We performed a "phage- ELISA" to confirm the interaction between the recombinantly expressed mIgE tail and the selected HAX-1-expressing phage. Phage, expressing the longer HAX-1-cDNA clone (231 C-terminal aa) or the shorter HAX-1-cDNA clone (213 C-terminal aa), were prepared, and the phage titer (PFU per milliliter) was measured. ELISA plates were coated with CH4-tail or CH4, respectively, and decreasing phage dilutions were applied. Signals obtained from binding to CH4 were subtracted from the CH4 tail signals as unspecific background. Shown are the longer HAX-1 cDNA clone (231 C-terminal aa) ({blacksquare}), the shorter cDNA clone (213 C-terminal aa) ({blacktriangleup}), and the control phage, encoding an unrelated protein ({circ}).

 
HAX-1 binds the IgE tail with moderate affinity

The association between the synthetically produced histidine (His)-tagged mIgE-tail and rHAX-1 was further tested by surface plasmon resonance analysis (SPRA) using the Biacore X device. Four thousand resonance units of rHis-tagged murine HAX-1 was coupled to flow cell 2 of a CM5 chip. The empty flow cell 1 served as reference. The synthesized IgE tail peptide was injected at different concentrations (5–100 µM) and the surface plasmon resonance was recorded. Data were analyzed with BIAevaluation software. We calculated an association constant (KA) of 1.4 x 104 M–1 and a dissociation constant (KD) of 7.4 x 10–5 M from the respective curves (Fig. 4). The calculated KD of 7.4 x 10–5 M indicated a biologically relevant association.


Figure 4
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  		FIGURE 4. Association and dissociation curves between HAX-1 and mIgE tail in SPRA. The rHAX-1 protein was immobilized on a CM5 chip, and the mIgE-tail peptide (concentrations from 5 to 100 µM) was used as soluble analyte. The KA = 1.4 x 104 1/M and KD = 7.4 x 10–5 M were calculated from the association and dissociation curves using the Langmuir binding model.

 
Endogenous HAX-1 coprecipitates with the recombinant mIgE tail in vitro

To investigate whether full-length HAX-1 interacts with the recombinant mIgE-tail, we performed coimmunoprecipitation experiments. The recombinant fusion protein of tail and carrier (CH4 tail) was added to the lysate of J558L myeloma cells (in vitro precipitation). As control, we performed the same precipitation with the carrier protein (CH4) alone. Anti-5xHis Ab was used to precipitate the His-tagged recombinant proteins. Endogenous murine HAX-1 was detectable by Western blot analysis only in the precipitate with the tail protein (CH4 tail) and not in samples precipitated with the carrier protein alone (Fig. 5).


Figure 5
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  		FIGURE 5. In vitro interaction of HAX-1 with the mIgE tail (in vitro coimmunoprecipitation). A total of 2 x 107 J558L (murine myeloma cell line) cells were lysed, and 30 µg of the carrier-tail protein (CH4 tail) or carrier protein (CH4) as negative control, respectively, were added to the lysates. The His-tagged CH4 tail and CH4 were precipitated with anti-5xHis Ab. After SDS-PAGE proteins were detected by immunoblotting. Upper panel, Developed with anti-5xHis Ab (CH4 tail and Ch4) and the lower panel with anti-HAX-1 mAb (endogenous HAX-1). The lysates (lys) (in lanes 1 and 2) show the carrier-tail protein (CH4 tail) or carrier protein (CH4), respectively, and the endogenous HAX-1 expression. The precipitates (prec) are shown in lanes 3 and 4. HAX-1 coprecipitates with CH4 tail but not with CH4.

 
HAX-1 coprecipitates with mIgE in the cell line JW813/4 BCR+

To investigate whether full-length HAX-1 interacts with the endogenously expressed mIgE tail, we used the JW813/4 BCR+ cell line. This cell line was stably transfected with a mIgE H chain construct that associated with the endogenous {lambda} L chain to form an NP-specific BCR of class IgE on the surface of the plasma membrane (1). This mIgE molecule was precipitated with NP-Sepharose. To exclude any BCR-mediated signaling and thus any de novo phosphorylation events, the specific Ag (NP-Sepharose) was added to the chilled and cleared cell lysates. As control, we performed a precipitation with empty (CL-2B) Sepharose. Endogenous HAX-1 coprecipitated with mIgE but could not be detected in the control precipitation. As additional controls, we did the same precipitations with JW813/4 BCR– cells. HAX-1 was not detectable in any of these control precipitations (Fig. 6).


Figure 6
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  		FIGURE 6. In vivo interaction of HAX-1 with the mIgE-tail (in vivo coimmunoprecipitation). HAX-1 and the endogenous mIgE were coprecipitated form a murine B cell line expressing NP-specific mIgE (JW813/4 BCR+). The mIgE was precipitated with NP-conjugated Sepharose (NP). Unconjugated Sepharose (CL-2B) was used as negative control. As second negative control, we performed all precipitations also in the JW813/4 BCR– cell line, which does not express mIgE. Upper panel, Developed with anti-murine IgE polyclonal rat serum and the lower panel with anti-HAX-1 mAb. The lysates (lys) (in lanes 1 and 2) show the endogenous HAX-1 expression in the JW813/4 cells with and without mIgE H chain, designated as BCR+ and BCR–, respectively. Lanes 3–6 show the precipitates (prec) with either NP-Sepharose or control (CL-2B)-Sepharose of these two cell lines. Lane 8 in the upper panel shows the position of the IgE band obtained with an IgE standard (arrow). HAX-1 coprecipitates with mIgE and was not detected in any of the negative controls.

 
siRNA-based knock down of HAX-1 in the B cell line JW813/4 BCR+

To elucidate a biological function of HAX-1 in the mIgE+ B cell line JW813/4 BCR+, we produced a HAX-1 knockdown by RNA interference technology using siRNA. JW813/4 BCR+ cells were transfected with pU6-siRNA expression vectors for RNA interference with HAX-1 expression. The two siRNA plasmids encoding two different HAX-1 siRNA targets led to the expression of short stem-loop RNAs homologous to 21 (target 1) or 19 (target 8) nucleotides of the murine HAX-1 mRNA. The two HAX-1 targets were transfected and after transfection single clones were isolated by limiting dilution. Stable integration of the U6 promoter and the siRNA target sequence was tested by PCR. Two PCR-positive clones per target were further analyzed by real-time PCR (Fig. 7A) and Western blot detection of HAX-1 (Fig. 7B), to test their ability to interfere with HAX-1 expression on the mRNA and on the protein level. The clones transfected with target 1 were designated C1 and D1, and the clones transfected with target 8 were designated A4 and D4. To monitor unspecific effects caused by siRNA-plasmid transfection, we additionally produced clones stably transfected with a control target without homology to HAX-1, designated B1 and B3. Fig. 7A shows that the two clones transfected with target 1 (C1 and D1) varied significantly in their HAX-1 expression. C1 reduced the HAX-1 mRNA level slightly but not significantly compared with wild-type JW813/4 BCR+ cells, whereas D1 led to a 48% reduction in mRNA amounts. The two clones transfected with target 8 (A4 and D4) led to a reduction of mRNA of 90 and 93%, respectively, compared with the wild type. The control target (clones B1 and B3) did not significantly influence HAX-1 mRNA expression.


Figure 7
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  		FIGURE 7. "Knockdown" of HAX-1 expression in JW813/4 BCR+ cells by RNA interference. A, Real-time PCR measurements of HAX-1 mRNA amounts in clones stably transfected with siRNA-expressing vectors. HAX-1-mRNA levels were normalized using Arpb mRNA expression levels. The fold change expresses the ratio of HAX-1 and Arpb mRNA levels in each sample. "C1 and D1" represent clones transfected with HAX-1-siRNA target 1; "A4 and D4" represent clones transfected with HAX-1-siRNA target 8; "B1 and B3" represent clones transfected with the control target; BCR+: wild-type JW813/4 expressing mIgE; BCR–: JW813/4 without mIgE. C1 shows no significant reduction of HAX-1 expression compared with wild-type or control targets. D1 shows a reduction of 48% and A4 and D4 show a 90 and 93% reduction. B, Immunoblots of crude cell lysates of JW813/4 BCR+ clones stably transfected with siRNA expression vectors (pU6-siRNA). HAX-1-protein (lower band, arrow); {alpha}-tubulin (upper band, arrow) was used as loading control. Immuoblots (B) correlate with real-time PCR measurements (A).

 
HAX-1 protein expression in the siRNA transfectants was assessed by Western blot detection. The protein expression levels (Fig. 7B) align with the results of the real-time PCR measurements.

HAX-1 protein levels influence Ag-internalization efficiency

HAX-1 is associated with the cytoskeleton (15) and it was suggested that HAX-1 participates in clathrin-mediated endocytosis of the surface receptor BSEP in Madin-Darby canine kidney II cells (22). Therefore, we asked whether the knockdown of HAX-1 expression in siRNA transfectants influenced the mIgE-mediated Ag internalization in JW813/4 BCR+.

The JW813/4 BCR+ transfectants with stable integrated HAX-1-siRNA targets were tested for their ability to internalize BCR after BCR stimulation with anti-IgE Ab. We found a correlation between HAX-1 expression levels and the ability of the respective clone to internalize the Ag. Clone C1 (target 1) showed no significant reduction of HAX-1 levels and in the internalization assay, we measured the highest internalization efficiency of all tested clones. Clone D1 (target 1) showed a 48% reduction of HAX-1 levels and in our BCR-internalization assay we measured approximately half the values of clone C1. The two clones transfected with target 8 (clones A4 and D4), showing a 90% reduction of HAX-1 expression, exhibited the lowest efficiency in internalization (Fig. 8). Table I demonstrates the correlation between HAX-1 levels and the internalization efficiency after 20 min. The internalization of clone C1 was set to 100% and the reduction relative to C1 was calculated for the other tested clones. For clone D1, a 48% reduction in HAX-1 protein levels led to a reduction of 48% in internalization efficiency. For target 8 (clones A4 and D4), this correlation is not so explicit and the reduction of 90% in HAX-1 protein levels led to a 53% reduction in internalization efficiency.


Figure 8
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  		FIGURE 8. BCR internalization in mIgE-expressing cell lines (JW813/4 BCR+) stably transfected with HAX-1 siRNA targets. IgE-BCR was stimulated with anti-IgE-FITC Ab (EM95-3-FITC) for the indicated time. FITC decreasing fluorescence is due to the pH shift upon internalization. The decrease of fluorescence over time was measured. One hundred percent fluorescence is the fluorescence at time "0" meaning 0% internalization. No fluorescence is calculated as 100% internalization. The reduction of HAX-1 protein levels led to a reduced efficiency of Ag internalization. Values were calculated from ten independent experiments. HAX-1-siRNA target 1 clone C1 ({diamond}), HAX-1-siRNA target 1 clone D1 (•) and HAX-1-siRNA target 8 (clones A4 and D4) ({blacktriangleup}) as indicated.

 

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  		Table I. Ag internalization after 20-min incubation correlates with HAX-1 mRNA levels

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we demonstrated in different independent experiments the direct interaction of murine HAX-1 with the cytoplasmic tail of mIgE. Using phage display technology, we isolated HAX-1-expressing phage clones and proved their specific binding to the recombinant mIgE tail in direct ELISA experiments (Fig. 3). Thus, explanations, which might favor a protein-protein-interaction, supported or bridged by a third protein (e.g., reported for Bcl-2-HAX-1-EBNA-LP (20)) could definitely be excluded. The calculated KD of 7.4 x 10–5 M (Fig. 4), using SPRA, indicated a biologically relevant association between HAX-1 and the cytoplasmic tail of mIgE. The biologically relevant interaction between HAX-1 and the mIgE tail was further demonstrated with independent coimmunoprecipitation experiments (Fig. 5: in vitro; Fig. 6: in vivo). Because of our performed experimental setup, we concluded that neither the in vitro nor the in vivo interaction between HAX-1 and mIgE depends on any de novo protein modification (i.e., phosphorylation). Finally, we measured the mIgE internalization efficiency in mIgE+ myeloma cells with reduced HAX-1 protein levels (Fig. 7) and clearly showed that reduction of HAX-1-protein levels reduced the internalization efficiency (Fig. 8).

Already before, Tarlinton (31) anticipated that truncation of the cytoplasmic tail of mIgG1 (32) and mIgE (33) would result in a defect in Ag presentation. Indeed, the YANIL motif inside the cytoplasmic tail of IgE (Fig. 9) matches the consensus sequence of cytoplasmic internalization motifs and reflects the (single)-core motif of an ITAM (YxxL/Ix6–8YxxL/I (34)). However, phosphorylation of Y622 in the cytoplasmic tail of IgE could never be shown. Nevertheless, the Y622ANIL motif seems to play an important role in the interaction with HAX-1. Human HAX-1 (73% homology and 69% identity with murine HAX-1) was shown to interact with the K15 protein of Kaposi’s sarcoma-associated herpes virus via a conserved YASIL motif within the C terminus of K15 (17). Due to the striking similarity between these two motifs, it seems very likely that murine HAX-1 interacts with the murine mIgE-tail via the YANIL-motif. Interestingly, as outlined in Fig. 9, the YANIL motif is not present in the cytoplasmic tails of IgG isotypes. This might indicate that the interaction between HAX-1 and the IgE cytoplasmic tail is specific for this isotype. This assumption, however, needs detailed experimental approach in the future.


Figure 9
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  		FIGURE 9. Sequence alignment of the cytoplasmic tails of murine Ig isotypes. Amino acid residues, not matching the IgE sequence are boxed. The YANIL motif is marked in gray.

 
To define a first biological function of the mIgE-HAX-1 interaction, we focused on an experimental approach describing the internalization capacity of mIgE with and without (or decreased) HAX-1 expression. Indeed, Ortiz et al. (22) showed presence of HAX-1 in clathrin-coated vesicles and HAX-1 participation in clathrin-mediated endocytosis, which itself depends on the functionality of cortactin. Cortactin is an F-actin-associated protein that localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction suggests that cortactin may participate in one or several endocytic processes. Cao et al. (35) suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. However, cortactin is not expressed in normal B lymphocytes or plasma cells (36), but Urono et al. (37) suggested that in hemopoietic cells, HS1 performs a cortactin-homologous function by modulating the actin assembly. The structure of HS1 is closely related to that of cortactin although HS1 contains only 3.5 repeat units, in contrast to 5.5 or 6.5 repeat units found in cortactin isoforms. Urono et al. (37) showed that HS1 is able to bind to F-actin with modest affinity and furthermore, that HS1 is able to interact directly with actin-related protein (Arp)2/3 complex and activate Arp2/3 complex-mediated actin nucleation and branching in a manner similar to cortactin. Thus it seems very likely that hemopoietic cells encompass a unique actin-polymerization system that involves HS1 and the Arp2/3 complex.

Interestingly, HAX-1 is a binding partner of both, cortactin and HS1, which explains our interest in the influence of HAX-1 on receptor-mediated internalization of Ag. Indeed, HAX-1-depletion retarded BCR internalization (Fig. 8). A 48% reduction of HAX-1 level led to a 48% reduction of internalization efficiency and a 90% reduction of HAX-1 levels reduced the internalization efficiency by 53%. From our point of view, the reduced internalization efficiency in answer to HAX-1 reduction explains the phenotypes of our knockout mouse strain IgEKVK{Delta}tail, expressing a truncated cytoplasmic tail of three amino acids (K-V-K). The truncation of the tail resulted in a 50% decrease of serum IgE, a weak secondary response, linked with a missing affinity maturation for IgE Abs (38). At that time, we concluded that the reduced IgE titers solely reflect the loss of biological activities associated with the cytoplasmic domain of IgE. Two hypotheses could be brought forward. First, signals generated via mIg are needed at all times, not only for the maturation process but also for the expansion of Ag-specific cells. Second, Ag presentation to Th cells is necessary during an Ab response, and only the Ag receptor is capable of effective Ag capture for presentation (33, 38). In the present study, we suggest that the incapability of the truncated tail to bind HAX-1 and therewith linked decreased Ag receptor internalization efficiency is responsible for the observed decreased serum IgE levels in IgEKVK{Delta}tail.

Summarizing, we suggest a model in which HAX-1 physically links the mIgE molecule to the cytoskeleton via its interaction with HS1. This link would be crucial for the efficient internalization of Ag in mIgE+ B cells. In B cells with a truncated mIgE tail (33), BCR internalization would be less efficient due to the absence of this link resulting in decreased Ag presentation to T cells. Especially in germinal centers, where B cells undergo rapid proliferation, it might be necessary to achieve rapid peptide loading to get T cell help in every cell cycle. This model is also in agreement with the fact that tail-knockout mice (33) show defects in the selection of high-affinity Abs in the hypermutation process in germinal centers (38). In these knockout mice, even hypermutated B cells that did retain the specificity for the Ag carrying the respective T cell epitope would not get sufficient specific T cells support for repeated rounds of positive selection.

From the theoretical point of view, cytoplasmic tails of mIgs might commit signals transmitted by 1) the tail alone, by 2) sterically influencing the binding of adaptor proteins to the Ig{alpha}-Igbeta coat proteins, or by 3) their capacity to actively bind proteins. With our current report, we clearly supported the hypothesis for the existence of actively cytoplasmic tail-binding proteins.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Experimental work was supported by the Austria Science Foundation Projects P-19017 and T166 (Hertha Firnberg) and the Austrian National Bank (OENB Grant 11710), the Swiss National Science Foundation (Grants 31-63382.00/2 and 310000-112540), and the OPO Foundation, Zürich. Back

2 Address correspondence and reprint request to Dr. Gernot Achatz, Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. E-mail address: gernot.achatz{at}sbg.ac.at Back

3 Abbreviations used in this paper: mIg, membrane-bound Ig; HAX-1, HS-1-associated protein X-1; EBNA, Epstein-Barr nuclear Ag; DHFR, dihydrofolate reductase; CH, constant region H chain; Arp, actin-related protein; NP, nitrophenyl; siRNA, small-interfering RNA; His, histidine; fwd, forward; rev, reverse; SPRA, surface plasmon resonance analysis. Back

Received for publication March 1, 2006. Accepted for publication April 26, 2006.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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