Glycosylation and Size of IgA1 Are Essential for Interaction with Mesangial Transferrin Receptor in IgA Nephropathy
Ivan C. Moura*,
Michelle Arcos-Fajardo*,
Charlotte Sadaka*,
Valérie Leroy*,,
Marc Benhamou*,
Jan Novak,
François Vrtovsnik,
Elie Haddad*,,
Koteswara R. Chintalacharuvu|| and
Renato C. Monteiro*
*INSERM E-0225, Bichat Medical School, Paris, France; Pediatric Nephrology Unit, Robert-Debré Hospital, Paris, France; Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama; Bichat-Claude Bernard Hospital, Paris, France; and ||Department of Microbiology, Immunology and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles, California
Correspondence to Dr. Renato C. Monteiro, Bichat Medical School, 16, rue Henri Huchard, 75018 Paris, France. Phone: 00-33-1-44-85-62-61; Fax: 00-33-1-44-85-62-60; E-mail: monteiro{at}bichat.inserm.fr
ABSTRACT. Transferrin receptor (TfR) has been identified asa candidate IgA1 receptor expressed on human mesangial cells(HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangialIgA1 deposits, and is overexpressed in patients with IgA nephropathy(IgAN). Here, structural requirements of IgA1 for its interactionwith mesangial TfR were analyzed. Polymeric but not monomericIgA1 interacted with TfR on cultured HMC and mediates internalization.IgA1 binding was significantly inhibited (>50%) by solubleforms of both TfR1 and TfR2, confirming that TfR serves as mesangialIgA1 receptor. Hypogalactosylated serum IgA1 from patients withIgAN bound TfR more efficiently than IgA1 from healthy individuals.Serum IgA immune complexes from patients with IgAN containingaberrantly glycosylated IgA1 bound more avidly to TfR than thosefrom normal individuals. This binding was significantly inhibitedby soluble TfR, highlighting the role of TfR in mesangial IgA1deposition. For addressing the potential role of glycosylationsites in IgA1-TfR interaction, a variety of recombinant dimericIgA1 molecules were used in binding studies on TfR with Daudicells that express only TfR as IgA receptor. Deletion of eitherN- or O-linked glycosylation sites abrogated IgA1 binding toTfR, suggesting that sugars are essential for IgA1 binding.However, sialidase and -galactosidase treatment of IgA1 significantlyenhanced IgA1/TfR interaction. These results indicate that aberrantglycosylation of IgA1 as well as immune complex formation constituteessential factors favoring mesangial TfR–IgA1 interactionas initial steps in IgAN pathogenesis.
IgA nephropathy (IgAN), the most common glomerulonephritis anda major cause of renal failure worldwide, is characterized bythe presence of IgA1 deposits in the mesangium (1, 2). The mechanisminvolved in mesangial deposits and proliferation, characteristicsof the initial phase of IgAN, is a major issue in understandingthe development of this disease. Enhanced (two- to threefold)levels of serum IgA observed in patients with IgAN cannot fullyexplain IgA deposition, because other IgA-associated diseases,such as AIDS and IgA myeloma, also have increased levels ofplasma IgA that do not always result in IgAN. An extrarenalorigin of IgAN is suggested by recurrence of IgA1 deposits afterrenal transplantation, indicating that circulating factors arecrucial (3, 4). It is now accepted that structural abnormalitiesof IgA1 are associated with the disease development (4). Patientswith IgAN display two major alterations in the IgA system: (1)increased levels of circulating IgA-containing immune complexes(5–7) and (2) the generation of abnormally glycosylatedIgA1 (8). IgA1 has heterogeneous O-glycans moieties in its hingeregion (three to five O-linked carbohydrate chains). In IgAN,these O-glycans can be found incompletely galactosylated (7).
Several mechanisms may be responsible for the formation of IgA1-containingimmune complexes in IgAN. One of the proposed mechanisms isbased on the presence of hypogalactosylated IgA1 in serum thatexposes terminal N-acetyl-D-galactosamine (GalNAc) generatingneo-epitopes that are recognized by naturally occurring IgGand IgA1 antibodies, inducing circulating immune complexes (CIC)(8). The presence of abnormal O-glycans is also related to IgA1stability in solution, as these molecules are prone to autoaggregation(9). Another mechanism implicated in IgA-complex formation isbased on the enhanced binding of IgA1 from sera of patientswith IgAN to FcRI (10) that induces the release of a solubleform of FcRI (Mr 50 to 70 kD). The pathogenic role of thesesoluble FcRI-IgA1 complexes was demonstrated by transfer experimentsusing mice transgenic for the human FcRI that spontaneouslydevelop IgAN (11). However, other forms of soluble FcRI (Mr30 kD) exists in normal individuals and are not involved inIgAN (12). Finally, sera of patients with IgAN may contain avariety of CIC that involve extracellular matrix proteins suchas fibronectin and collagen (4, 13, 14).
IgA1 and IgA1-containing immune complexes binding to human mesangialcells (HMC) seem mediated by an IgA receptor(s) (15). IgA1 canbind to rat and human HMC in a dose-dependent manner, and thisleads to IgA internalization and degradation (15–21).Binding of heat-aggregated IgA to HMC induces cell activation,mobilization of intracellular calcium storage, protein phosphorylation(22), and release of IL-6 and TNF- (16). However, none of theknown IgA receptors (CD89, the asialoglycoprotein receptor,and the polymeric Ig receptor) is expressed by HMC (15, 18–21, 23, 24). Recently, we reported that the transferrin receptor(CD71/TfR) could serve as a mesangial IgA1 receptor. Indeed,TfR binds IgA1 but not IgA2 (25), and its expression is enhancedin the mesangium of patients with IgAN and co-localizes withIgA1 deposits (25, 26).
In this study, we analyzed the role of glycosylation and themolecular form of IgA in the interactions with TfR expressedon HMC. Our results revealed that polymeric IgA1 but not monomericIgA1 binds to TfR and that either TfR1 or TfR2 can participatein this binding. We showed that IgA1 isolated from serum ofpatients with IgAN and macromolecular IgA containing hypogalactosylatedIgA1 present in serum from patients with IgAN showed enhancedbinding to TfR expressed on HMC. Finally, we analyzed the roleof IgA1 glycosylation in TfR binding by studying either theinteraction of a variety of recombinant IgA1 protein lackingN-linked or O-linked glycosylation sites or the ability of deglycosylatedIgA1 to bind TfR. All together, our data indicate that despitethat O- and N-linked glycosylation sites of IgA1 are essentials,hypogalactosylation or desialylation of IgA1 promotes enhancedIgA1 binding to TfR.
Subjects
Sera from patients with IgAN (diagnosed by the presence of predominantIgA1 deposits in the mesangium associated with focal or diffusemesangial cell proliferation; n = 15) and healthy, randomlyselected subjects (n = 15) were studied. In each instance, informedconsent was obtained from the donors for the use of blood samplesfor experimental purposes.
Antibodies and Reagents
The following mAb were used: A24 anti-TfR (2b) (25), MA712 anti-TfR(2a; Pharmingen, San Diego, CA), CH-EB6-8 (1) anti-IgA (AmericanType Culture Collection, Rockville, MD) (27), 1-155-1 (3) anti-IgA1,and 14-3-26 (2b) anti-IgA2 (28). Polyclonal anti-human transferrin(Sigma Chemical Co., St. Louis, MO) was coupled to activatedSepharose 4B beads (Amersham Biosciences, Uppsala, Sweden) accordingto the manufacturer’s recommendations. Human myeloma IgA1(Dou) was purified as described (25). IgA2 (IgA2m[2]; Fel) wasprovided by Dr. J. Mestecky (University of Alabama at Birmingham,Birmingham, AL) and IgA2 (IgA2m[2] Bel) was a gift from Dr.P. Aucouturier (Inserm E0209, St. Antoine Hospital, Paris, France).Fractions containing monomeric (m) and polymeric (p) IgA wereprepared by size-exclusion chromatography on Superdex 200 columnsthrough HPLC (Amersham; >99% pure). Transferrin-free IgA(purified by gel filtration and immunoabsorption through anti-transferrinSepharose 4B columns) was biotinylated as described (25). Humanserum IgG was purified by ammonium sulfate precipitation andDEAE ion exchange chromatography. Proteins were quantified bythe BCA method according to the manufacturer’s instructions(Pierce, Rockford, IL).
The generation and purification of recombinant human IgA1 (1),IgA1 with the hinge region of IgA2 (1 with 2 hinge), IgA2 withthe hinge region of IgA1 (2 with 1 hinge), IgA1 lacking theN-linked glycosylation sites (1 with no N-CHO), and IgG1 havebeen previously described (29–31).
Modification of IgA glycans composition were obtained by enzymatictreatments of myeloma pIgA1 (Dou) using N-glycosidase F, O-glycosidase,neuraminidase from Vibrio cholerae (Roche Molecular Biochemicals,Mannheim, Germany), and -galactosidase from bovine testes (Sigma-Aldrich)in accordance with the manufacturer’s instructions andas described (8).
Cells
Primary HMC from single donors were purchased from a commercialsource (Clonetics, San Diego, CA). Cells were cultured in RPMI1640, supplemented with glutamine (2 mM), 5 µg/ml insulin,20% FCS (Life Technologies, Gaithersburg, MD), 7 mM glucose,50 U/ml penicillin, and 50 µg/ml streptomycin in a 7%CO2 atmosphere. Cells were detached with 0.25% trypsin and 0.5%EDTA (Invitrogen, Carlsbad, CA). Trypsinization was no longerthan 2 min at 37°C, and the reaction was stopped by additionof cold RPMI containing 10% FCS. Under these conditions, thecells were viable (>95%) and TfR expression was not affectedas evaluated by flow cytometry using an anti-TfR mAb. Studieswere performed using HMC from passages 4 to 8. Established Daudicell line was obtained from American Type Culture Collection.
Expression and Purification of TfR1 and TfR2 Ectodomains
Soluble (s) versions of human TfR1 and TfR2 were expressed ina lytic baculovirus/insect cell expression system as describedpreviously (32). Stability of soluble TfR was assessed by measuringits binding to holotransferrin. The construct for the irrelevantcontrol soluble form of HLA-DR4 was provided by J.M. Fourneau(Necker Hospital, Paris, France).
Affinity Purification of IgA1 Fractions
IgA1 fractions were purified by jacalin affinity and anti-IgA1chromatography. Briefly, jacalin was purified from jackfruitextracts by DEAE column as described previously (33) and coupledto Sepharose 4B beads (Pharmacia). Serum fractions were dilutedin PBS and absorbed on jacalin-Sepharose columns, and lectin-bindingproteins were eluted overnight with 0.5 M galactose as described(33). Serum IgA1 was also purified by affinity chromatographyusing anti-IgA1 Sepharose columns as described previously (8, 15, 34).
Immunofluorescence Analysis
Cells (0.25 x 106) were preincubated with 10 µl of humanIgG (10 mg/ml) for 15 min on ice to mask FcR. IgA binding wasexamined using an indirect immunofluorescence assay in whichcells that were preincubated with human IgG were incubated with10 µl of IgA (0.5 to 10 mg/ml) for 1 h on ice before washingand incubation with a biotinylated anti-IgA mAb (clone CH-EB6-8that recognizes both IgA1 and IgA2) for 20 min at 4°C. Afterwashes, Allophycocyanin-labeled streptavidin (Southern BiotechnologyAssociates, Birmingham, AL) was used as a developing reagent.In some experiments, biotinylated HPLC-purified IgA1 (free oftransferrin) plus phycoerythrin-labeled streptavidin were used.For inhibition studies, biotinylated myeloma IgA or unlabeledIgA-containing circulating immune complexes were preincubatedwith soluble receptors at indicated concentrations for 1 h beforeaddition to the cells. Immunofluorescence was finally analyzedby flow cytometry (FACScalibur; Becton Dickinson, NJ).
Confocal Microscopy
HMC were incubated for 30 min at 4°C to block membrane recyclingand then labeled with biotinylated pIgA1 (0.5 mg/ml) for 60min at 4°C. After washes, cells were incubated for 30 minwith streptavidin phycoerythrin (Southern Biotechnology Associates;1:50 dilution). Cells were washed and shifted to 37°C fordifferent time points. HMC were fixed in 4% paraformaldehyde,quenched with 0.1 M glycine, blocked in PBS containing 2% FCS,and permeabilized with 0.005% saponin in PBS containing 0.1%BSA. TfR next were stained with the mAb A24 (10 µg/ml)for 30 min at 4°C. After washes, A24 mAb was revealed bya goat anti-mouse IgG antibody conjugated to Cy-5 (Jackson ImmunoResearch,West Grove, PA). Slides were examined with a confocal lasermicroscope system (LSM 510 Carl Zeiss, Oberkochen, Germany).
Fractionation of Serum
A total of 0.5 ml of serum was diluted in phosphate buffer (0.05M Na2SO4, 0.02 M NaH2PO4 [pH 7.5]) (8), filtered, and separatedby gel filtration through an S-300 Sephacryl column (AmershamPharmacia) connected to an HPLC AKTA-basic automated liquidchromatography system (Amersham Pharmacia). The molecular sizeof each fraction was determined by the content of differentserum proteins revealed by immunoelectrophoresis. Fractionsof 1.0 ml were collected and analyzed. IgA concentration inthe fractions was determined using a sandwich ELISA method.
ELISA
For determining IgA concentration in different column fractions,plates were coated with mAb CH-EB6-8, anti-IgA1, or anti-IgA2mAb at 5 µg/ml in borate-buffered saline (BBS) for 2 hat room temperature. The wells were then washed twice in BBScontaining 0.05% Tween and blocked for 2 h at room temperaturein PBS containing 1% BSA and 0.1% sodium azide. Column fractions(diluted at 1:100) were incubated overnight at 4°C. A polyclonalanti-IgA alkaline phosphatase (AP) conjugate (Southern BiotechnologyAssociates) was used as developing antibody. The optical densityat 450 nm was measured after addition of streptavidin-AP substrate(Sigma) following the manufacturer’s instructions. SolubleTfR was measured in serum by ELISA using two mAb, clone MA712from Pharmingen and clone A24 (25).
Gel Electrophoresis and Immunoblotting
SDS-PAGE and Western blotting were performed as described elsewhere(35, 36). Briefly, 20 µg of protein extracts was solubilizedin SDS-sample buffer under nonreducing conditions at 100°C,subjected to electrophoresis in a 4% polyacrylamide gel, andelectroblotted onto polyvinylidene fluoride membranes (Millipore,Bedford, MA). Membranes were blocked overnight with TTBS buffer(50 mM Tris-HCl, 150 mM NaCl [pH 7.4], 0.05% Tween 20) containing4% BSA (wt/vol) at 4°C. For detecting IgA, strips were incubatedwith 1 µg/ml biotinylated anti-IgA mAb CH-EB6-8 in 1%BSA in TTBS. Alternatively, the presence of altered glycosylatedIgA was verified using biotinylated Helix aspersa (HAA; EY Laboratories)at 1 µg/ml using the same conditions described above.Membranes were washed and then incubated with streptavidin-horseradishperoxidase (Bio-Rad Laboratories, Hercules, CA) at a 1:30,000dilution in TTBS buffer containing 1% BSA. Blots were developedwith West-Pico substrate (Pierce) according to the manufacturer’sinstructions. Band intensity was evaluated by NIH Image softwareand considered as increased when greater than the mean + 2 SDof control bands intensity.
Statistical Analyses
The results were analyzed by independent sample two-tailed ttest. Results are presented as means ± SD.
Polymeric but not Monomeric IgA1 Binds to TfR on HMC
We have previously shown, using a direct immunofluorescenceassay based on biotinylated IgA preparations, that mIgA1 bindsmore than pIgA1 to TfR (25). Because our mIgA1 preparationscontained minimal (<1%) amounts of transferrin, we reexaminedthe type of IgA form involved in TfR binding using an indirectimmunofluorescence assay developed by a biotinylated anti-humanIgA mAb (clone CH-EB6-8 that recognizes both IgA1 and IgA2 subclasses)and streptavidin-Allophycocyanin. We evaluated the binding ofseveral types and forms of IgA, including mIgA1, pIgA1, mIgA2,pIgA2, and secretory IgA (SIgA) to HMC. HMC express TfR (25)but not the other known IgA receptors, including the CD89, thepolymeric-Ig receptor (pIgR), and the asialoglycoprotein receptor(ASGP-R) (15, 18–21, 23). Under our experimental conditions,only pIgA1 binding differed significantly from control humanIgG (Figure 1A). By contrast, using this method, we detectedbinding of both IgA1 and IgA2 to CD89 expressing cells (datanot shown). The sensitivity of this method was evaluated bya dose-response curve of IgA binding. Figure 1B shows FACS histogramsof a dose-response curve of pIgA1 binding. pIgA1 binding differsfrom the control IgG for values >0.5 mg/ml. Binding of pIgA1to HMC is linear, dose-dependent, and not saturable up to 10mg/ml (Figure 1C). The specificity of IgA1 binding to mesangialTfR was next verified by blocking mesangial binding of pIgA1by the addition of soluble forms of TfR1 and TfR2 (sTfR1 andsTfR2) produced in a baculovirus/insect cells expression system.Preincubation of pIgA1 with sTfR2 at a soluble receptor:pIgAmolar ratio of 1.5 inhibited by 55% the binding to HMC. Underthe same conditions, sTfR1 inhibited IgA1 binding by approximately50% (Figure 2A). The control soluble form of the unrelated HLA-DR4molecule did not inhibit IgA binding to these cells. In addition,inhibition of pIgA1 binding to HMC using both sTfR1 and sTfR2was dose dependent (Figure 2B). To address the specificity ofIgA1-TfR interaction, we performed similar experiments usingthe Daudi cell line, cells that express TfR as the only IgA1receptor (25). Both sTfR1 and sTfR2 at a soluble receptor:pIgAmolar ratio of 1.5 inhibited pIgA1 binding (Figure 2C). Usinganti-IgA immunofluorescence indirect assay, holotransferrinfailed to inhibit pIgA1 binding to Daudi and HMC up to 1 mg/ml(data not shown). These results indicate that pIgA1 bindingto HMC is mainly mediated by the transferrin receptor.
Figure 1. Polymeric IgA1 (pIgA1) but not monomeric IgA1 (mIgA1) binds to human mesangial cells (HMC). (A) Comparative immunofluorescence analysis of several types and forms of IgA including mIgA1, pIgA1, mIgA2, and pIgA2, and secretory IgA (SIgA) at 1 mg/ml. Human IgG (filled histogram) was used as negative control. IgA binding to HMC was evaluated using biotinylated anti-human IgA mAb (CH-EB6-8) and streptavidin-Allophycocyanin (APC) as the developing reagent. (B et C) pIgA1 binding is concentration dependent. (B) HMC were incubated with serial dilutions of pIgA1 and binding was detected by biotinylated CH-EB6–8 mAb followed by streptavidin-APC. (C) Fluorescence median intensities of FACS analyses of pIgA1 binding were plotted, and IgG was used as negative control.
Figure 2. pIgA1 binding to HMC is inhibited by transferrin receptor (TfR) ectodomains. (A) Immunofluorescence analysis of the ability of the TfR1, TfR2, and HLA-DR4 ectodomains to block biotinylated pIgA1 binding on HMC. Biotinylated pIgA1 was purified by gel filtration through HPLC using Sephacryl S-200 columns and further passed through anti-transferrin beads to eliminate any residual transferrin. pIgA1 fractions (99.9% pure) devoided of were then used for binding experiments. In each panel, the heavy dark line indicates the control pIgA1 binding and the thin line indicates binding in the presence of the indicated soluble protein. Human IgG was used as a negative control of binding (filled histogram). (B) Soluble (s) forms of TfR1 and TfR2 inhibit pIgA1 binding to HMC in a dose-dependent manner. The results are the mean inhibition (± SD) of median fluorescence intensity over background of FACS analysis of three independent experiments performed as in A. (C) Soluble (s) forms of TfR1 and TfR2 inhibit pIgA1 binding to Daudi cells. Experiments were performed as in (B) with Daudi cells.
pIgA1 Induces TfR Endocytosis and Co-localizes with Intracellular TfR Compartments
The transferrin receptor plays a key role in Fe uptake and isconstantly recycled in the cell (37). This homodimeric moleculebinds two Fe-loaded (holo) transferrin molecules at the cellsurface and is internalized. Recycling vesicles containing theTfR:holotransferrin are acidified, and Fe dissociates from Tf.The remaining Tf-TfR complex remains associated at pH 6.4 anddissociates after recycling to the cell surface at pH 7.4 (37).Under physiologic conditions, no internalization of TfR is observed.However, this can be achieved by anti-TfR antibodies. FunctionalIgA receptors such as CD89 and the ASGP-R are internalized anddegraded after binding of IgA-containing immune complexes (23).Therefore, we examined whether pIgA1 binding to TfR could mediateTfR endocytosis.
Cells stained with pIgA1 were allowed to endocytose, fixed,and stained intracellularly with the anti-TfR mAb A24. As shownin Figure 3, TfR was initially localized near the cell surfacein recycling vesicles, which is a characteristic feature ofTfR (37). After 5 min, pIgA1 co-localized with TfR at the cellmembrane and in the recycling vesicles. After 15 min, TfR wasfound in intracellular compartments, where it co-localized withinternalized pIgA1, indicating that pIgA1 induced endocytosisof TfR.
Figure 3. pIgA1 induces TfR endocytosis. Cell suspensions of HMC were incubated with 0.5 mg/ml biotinylated pIgA1 and further decorated with streptavidin-phycoerythrin (red) at 4°C. After washing, cells were shifted to 37°C for the time indicated. Cells were then washed, fixed, and permeabilized. TfR was then labeled with the mAb A24 and an anti-mouse-Cy-5 (blue).
Binding of IgA Immune Complexes from Sera of Patients with IgAN to Cultured Mesangial Cells Is Mediated by the TfR
To determine the binding characteristics of immune complexesfrom patients with IgAN to cultured mesangial cells, we comparedbinding characteristics of macromolecular IgA purified fromnormal subjects and patients with IgAN with mesangial TfR. Serafrom healthy control subjects and from patients with IgAN werefractionated using HPLC with a gel filtration column. Figure 4Ashows a representative profile of resolution through gelfiltration of normal and IgAN serum. Differences between theprotein profiles of sera from control subjects and patientswith IgAN occurred for high-molecular-weight (>850 kD) proteins.IgA distribution between these fractions was determined usingan ELISA capture assay (Figure 4B). The amount of macromolecularIgA species was increased in sera from patients with IgAN. Thepolymeric nature of IgA was further confirmed by anti-IgA Westernblot. Different fractions were separated in a nonreducing 4%SDS-PAGE and immunoblotted, and IgA was detected using a biotinylatedmonoclonal anti-IgA antibody (CH-EB6-8). IgA1 was predominantwithin these fractions, but a similar IgA1/IgA2 ratio was observedbetween control and patient fractions (not shown). As shownin Figure 4C, IgA from patients contained higher molecular weightpIgA than those from normal subjects. For addressing the questionof whether IgA from patients contained abnormal O-glycans, thesame protein amount of IgA-containing high-molecular-weightfractions from a patient and from a control subject were blottedwith biotinylated HAA (a lectin that binds specifically to terminalGalNAc). Enhanced HAA binding was observed only in the polymericIgA fractions from the patients with IgAN (Figure 4D).
Figure 4. Purification of IgA complexes from patients with IgA nephropathy (IgAN) and healthy subjects. (A) Serum from patients with IgAN and healthy subjects were fractionated by gel filtration through HPLC using a Sephacryl S-300 column. Separated fractions were analyzed for both their protein (A) and IgA (B) content. Molecular masses of the calibrated column are indicated at the top of the figure. (C) Detection of different IgA sizes contained in each fraction by immunoblotting using biotinylated anti-IgA and streptavidin–horseradish peroxidase (HRP) as a developing reagent. Molecular weight markers are indicated in the right side. (D) Detection of abnormally glycosylated IgA in high-molecular-size fractions by immunoblotting using biotinylated-HAA and streptavidin-HRP as developing reagent.
We next addressed the characteristics of macromolecular IgAbinding to mesangial TfR. The concentrations of macromolecularIgA from normal individuals and patients with IgAN were normalized,and these IgA preparations were incubated with HMC followedby FACS analysis of IgA binding. Under these conditions, threeof five samples of macromolecular IgA from sera of patientswith IgAN showed increased binding to HMC (Figure 5A). The bindingof macromolecular IgA to HMC was shown to be dose dependent(Figure 5B). These results confirm the data previously obtainedby others ((15) showing that high-molecular-weight IgA fromsera of patients with IgAN have enhanced binding to HMC. Thespecificity of IgA complexes binding to mesangial TfR was verifiedby inhibition of IgA binding to HMC by soluble forms of TfR1and TfR2; sTfR1 and sTfR2 significantly inhibited IgA complexbinding to HMC, whereas soluble HLA-DR4 did not (Figure 5C).Fractions containing low Mr IgA species (160 and 60-kD heavychain) did not bind TfR (not shown).
Figure 5. Binding of IgA-complexes from patients with IgAN to HMC is mediated by TfR. (A) HMC were incubated with 20 µg of IgA complexes (column fractions 22 to 25) from patients with IgAN and control subjects, and IgA binding to TfR was evaluated using biotinylated anti-human IgA mAB (CH-EB6-8) and streptavidin-APC as the developing reagent. (B) IgA complexes binding to HMC is concentration dependent. HMC were incubated with the indicated concentrations of IgA complexes from patients with IgAN, and IgA binding was evaluated by FACS analysis using biotinylated CH-EB6-8 mAb followed by streptavidin-APC. Median fluorescence intensities were plotted after subtracting values of each corresponding concentration of biotinylated IgG. (C) Soluble forms of TfR1 and TfR2 but not HLA-DR4 inhibit IgA complexes binding to HMC. IgA complexes were preincubated with soluble receptor at a 1.5 molar ratio. Complexes were then transferred to HMC, and IgA binding was evaluated as described above. Histograms represent mean ± SEM of three independent experiments.
Aberrantly Glycosylated IgA1 Binds More to TfR than Normal IgA1
Because IgA1 from sera of patients with IgAN had been shownto have altered O-linked glycosylation (7), we examined whetherpatient IgA1 has an enhanced binding capacity to TfR. IgA1 proteinswere isolated from sera of healthy donors and patients withIgAN using a jacalin-Sepharose affinity column (abnormally glycosylatedIgA can bind to some preparations of jacalin as reported (38)).The aberrantly glycosylated IgA was detected by HAA binding.IgA fractions were normalized in their protein content and blottedusing biotinylated HAA. Samples (seven of 15) from patientswith IgAN had an enhanced HAA binding, indicating the presenceof galactose-deficient IgA1 (data not shown). We therefore evaluatedthe ability of jacalin-purified IgA1 from patients with IgANand control subjects to bind to HMC (0.5 mg/ml). A significantincrease in IgA1 binding was observed with material from patientswith IgAN (Figure 6A).
Figure 6. Enhanced binding of jacalin-purified IgA from IgAN sera to HMC and Daudi cells. (A) Comparative immunofluorescence analyses of binding of jacalin-purified IgA from 12 patients with IgAN and 12 control subjects (20 µg each) to HMC. (B) Binding of jacalin-purified IgA from patients with IgAN and control subjects to Daudi cells. Binding was evaluated by CH-EB6-8 mAb followed by streptavidin-APC. Median fluorescence intensities were plotted after subtracting values of each corresponding concentration of control IgG (mean ± SD). (C) Affinity-purified IgA1 from patients with IgAN bind more to HMC than controls. IgA1 from patients and control subjects were purified by affinity chromatography, and 20 µg of each was incubated with Daudi cells. IgA binding to Daudi cells was evaluated using biotinylated anti-human IgA mAb (CH-EB6-8) and streptavidin-APC as the developing reagent.
Daudi cells express TfR as the only IgA receptor (the only IgAreceptor recovered from these cells by IgA affinity columnsis the TfR) (25). Therefore, we next studied binding characteristicsof patients’ IgA1 to these cells as compared with controlIgA. Figure 6B shows that jacalin-purified IgA1 from patientswith IgAN at the same concentration (0.5 mg/ml) bound significantlymore to Daudi cells than those from control subjects, confirmingdata obtained with HMC. Immuno-affinity purified IgA1 from bothcontrol subjects and patients with IgAN were also used in bindingexperiments on Daudi cells. Figure 6C shows that patient IgA1have an enhanced binding to Daudi cells.
IgA1 Binding to TfR Is Dependent on the Presence of Both O- and N-Linked Glycans
Because aberrantly glycosylated O-glycans in the hinge regionof IgA1 have been shown to be important in binding to mesangialcells in IgAN (15), we hypothesized that the O-linked carbohydratesin the hinge region of IgA1 could mediate the binding of IgA1to TfR on HMC. To test this hypothesis, we studied the bindingto TfR on Daudi cells of well-characterized recombinant IgA1,IgA2, IgA1 with IgA2 hinge region, IgA2 with IgA1 hinge region,and IgA1 lacking the N-linked glycosylation sites in the heavychains (31) (Figure 7A). The human IgA1 contains three to fiveO-linked glycans in a 26–amino acid hinge region locatedbetween domains C1 and C2, whereas IgA2, which has a 13–aminoacid hinge region, lacks the O-linked glycans (7, 30) (Figure 7A).All proteins contain predominantly polymers with J chain(data not shown). Significant binding to TfR was observed onlywith wild-type IgA1. However, this binding was lower than thatobserved for polymeric myeloma IgA1 (Dou; Figure 7, B and C).IgA2 did not bind to TfR on Daudi cells, suggesting that theIgA1 hinge region is required for binding to TfR. In addition,IgA2 containing the IgA1 hinge region did not bind to TfR, suggestingthat IgA1 hinge region is not sufficient to promote binding.Remarkably, IgA1 either lacking N-linked glycosylation in the heavy chains or lacking O-linked glycosylation did not bindto Daudi cells, indicating that both N- and O-linked glycosylationis required for IgA1 binding to TfR (Figure 7, B and C). Takentogether, these results suggest that both O- and N-linked glycosylationin the context of IgA1 is required for building the IgA1 bindingsite to TfR.
Figure 7. N- and O-glycosylation of IgA is important in IgA1/TfR interaction. (A) Schematic diagram of the H chain genes used to produce the recombinant antibodies. The exons are indicated by the rectangular boxes. Note that whereas the box between CH1 and CH2 codes for the of 1 hinge region, the box at the 5' end of CH2 in codes for the hinge region. The open diamonds over the exons s show the position of N-linked glycosylation sites. The circles in the hinge of IgA1 represent the O-linked glycosylation sites. (B) Comparative immunofluorescence analyses of binding of different myeloma pIgA (pIgAdou), wild-type recombinant dimeric IgA1(1), IgA1 with the hinge region of IgA2 (1 with 2 hinge), IgA2 with the hinge region of IgA1 (2 with 1 hinge), IgA1 lacking the N-linked glycosylation sites (1 with no N-CHO), and IgG1 have been previously described on Daudi cells. Cells preincubated with human IgG to mask FcR were incubated with different IgA, and binding was evaluated by CH-EB6-8 anti-IgA mAb followed by streptavidin-APC. Human IgG (hIgG) was used as a negative control of binding (dashed line). (C) Median fluorescence intensities were plotted after subtracting values of each corresponding concentration of control IgG (mean ± SD; n = 3).
Degalactosylation and Desialylation of IgA1 Enhances Its Binding Capability to TfR on Daudi and HMC
Altered glycosylated IgA present in the circulation of patientswith IgAN have an enhanced affinity to mesangial cells (15, 17–19, 21). However, whether those IgA glycoforms arecapable of interacting with TfR is unknown. Carbohydrate moietiesof IgA were modified using different enzymatic treatments, andIgA1/TfR interaction was analyzed on Daudi cells that expressthe TfR as the only IgA receptor (25). Figure 8A shows thatO-glycosidase abrogates IgA1 binding to TfR, whereas N-glycosidasehas no major effect. By contrast, neuraminidase and neuraminidaseplus -galactosidase significantly enhanced IgA1 binding to TfR.Desialylated and degalactosylated IgA1 also have an enhancedbinding to mesangial cells (Figure 8B). This enhanced bindingwas strongly inhibited by TfR ectodomains, indicating that TfRis mediating the interaction between aberrant O-linked glycosylatedIgA1 and HMC (Figure 8B).
Figure 8. Desialylation and degalactosylation of IgA1 enhances its binding capacity to TfR. (A) Median fluorescence intensities were plotted (mean ± SD; n = 3) of binding of myeloma pIgA1 and of desialylated or deglycosylated IgA1 on Daudi cells as indicated. IgG was used as a negative control. Cells preincubated with human IgG to mask FcR were incubated with different IgA, and binding was evaluated by CH-EB6-8 anti-IgA mAb followed by streptavidin-APC; *P < 0.05. (B) Comparative immunofluorescence analyses of binding properties of myeloma pIgA1 and of desialylated and degalactosylated IgA1 on HMC as indicated. The interaction to TfR was evaluated by using sTfR1 as a specific inhibitor.
Levels of Soluble TfR Are not Enhanced in Serum of IgAN Patients
Human serum usually contains a truncated form of the TfR thatcirculates in the blood complexed to transferrin (39). To investigatewhether IgA1 binding to TfR could induce secretion of the solubleTfR, we determined the levels of naturally occurring sTfR inserum. No significant differences were observed between controlsubjects and patients with IgAN (not shown). Therefore, thesedata indicate that IgA1 from patients with IgAN do not inducerelease of sTfR.
Fc receptors are involved in the etiopathogenesis of many glomerulonephritidesvarying from autoimmune to those of systemic origin (e.g., lupusnephritis, Goodpasture syndrome, IgAN) (23, 40). In IgAN, theidentity of an IgA receptor mediating the mesangial IgA1 depositionand cellular activation has been enigmatic for many years (4).Indeed, although the interaction of IgA1 with HMC has been demonstratedby several groups (15, 17–19, 21), the identity of thisputative mesangial IgA1 receptor has been elusive. Despite thepresence of aberrantly glycosylated IgA1 in IgAN, the ASGP-R,one of the most important receptors responsible for catabolismof proteins with terminal Gal and GalNAc, is not expressed onmesangial cells (15, 20) and neither are other IgA receptorssuch as CD89 and pIgR (15, 19, 20). Recently, we characterizeda novel mesangial IgA1 receptor, the TfR or CD71 (25). Thisreceptor is the only IgA receptor so far identified at the membraneof HMC. Its importance is underscored by the fact that in IgAN,TfR expression is enhanced in the mesangium and co-localizeswith IgA deposits (25, 26).
In this study, we first analyzed the interaction between differentmolecular forms of IgA and TfR expressed on cultured HMC. Usingan indirect immunofluorescence assay with an anti-IgA mAb, weshowed that polymeric but not monomeric IgA could bind to TfRexpressed on mesangial cells and to other cells that have IgAreceptor expression restricted to TfR, such as the Daudi cellline (data not shown).
Furthermore, we showed that IgA1 but not IgA2 binds the TfRon HMC and Daudi cells. However, these results showing no IgA2binding to HMC are in contrast with previous observations usingdirect measurements of radiolabeled IgA2 binding (15). Thisdiscrepancy could be due to a higher sensitivity of the latterassay than the indirect immunofluorescence technique used here.The observations that mesangial IgA deposits in IgAN are mainlycomposed of IgA1 polymers and that TfR is overexpressed in themesangium of patients with IgAN and co-localizes with IgA1 deposits(26) suggest that the TfR may be involved in binding of pIgA1to HMC.
The specificity of IgA1-TfR interaction was confirmed by theinhibition of IgA binding to HMC by recombinant TfR solubleforms. As both TfR1 and TfR2 ectodomains could block IgA1 bindingto HMC, we postulate that the TfR domain involved in IgA bindingis shared between these two receptors. This could be explainedby the significant homology (45% amino acid identity) observedbetween these proteins (41). TfR2 differs from TfR1 only bya shorter side chain encoded by a separated gene (42), and TfR2was shown to bind transferrin with low affinity and to mediateiron uptake (43). Our confocal data indicate that pIgA1 caninduce TfR endocytosis in HMC with accumulation in intracellularvesicles from 15 to 60 min of endocytosis. One can propose thatrecycling of TfR can amplify mesangial IgA complex deposition,thus favoring subsequent activatory mechanisms that could leadto mesangial activation in IgAN (43). The intracellular localizationof IgA in TfR-positive vesicles suggests that both moleculesare targeted together to endolysosomal vesicles where pIgA aredegraded. Whether both types of TfR are expressed by HMC andcould play a role in IgA1-mediated signaling and/or endocytosisremains to be determined.
IgA1 mesangial deposits have been reported to derive from CIC(7). The data presented here reveal an increased capabilityfor macromolecular IgA from patients with IgAN to interact withthe TfR expressed on HMC. These findings point to a role ofthe molecular size of IgA in the interaction between CIC andmesangial TfR, refining observations recently reported by others(15). The specificity of macromolecular IgA/TfR interactionon HMC was also shown by using soluble forms of TfR1 and TfR2as inhibitors. Both soluble forms of TfR induced approximately50% inhibition of IgA binding to cell membrane. The enhancementof IgA1 binding to TfR only when IgA is present in IgA-complexform indicates that IgA1 interaction with the TfR is of a verylow affinity and that its binding can be modulated by increasedavidity after polymerization or aggregation. The failure toblock completely IgA binding to HMC could be due to a loweraffinity/avidity of soluble versus membrane TfR or could indicatethat a second type of as-yet-uncharacterized mesangial IgA receptormay exist. A candidate molecule for such a second mesangialIgA receptor is the recently cloned Fc/µ receptor, a newFc receptor for IgA and IgM (44). Indeed, recently, transcriptsof Fc/µ receptor were described in mesangial cells thatwere upregulated after stimulation by proinflammatory cytokinessuch as IL-1 and TNF- (45). Fc/µ receptor contains onlyone Ig-like domain that is homologous to the first Ig-like domainof pIgR involved in IgA/IgM binding. However, this receptordoes not seem to account for IgA binding in quiescent HMC becauseIgM cannot inhibit IgA binding to HMC (21). Furthermore, weproduced recombinant ectodomain of Fc/µ receptor in baculovirus/insectcell system, and this protein failed to inhibit IgA1 bindingto HMC (data not shown). Although IgA interaction with the humanFc/µ receptor on mesangial cells has not yet been demonstrated,a putative role of Fc/µ receptor cannot be excluded duringinflammation.
Several authors have postulated that aberrant glycosylationof the hinge region of IgA1 is involved in formation of immunecomplexes or autoaggregation that may promote formation of mesangialIgA1 deposits (7). Aberrant patterns of glycans, such as increasedexposure of GalNAc residues and decreased content of Gal, havebeen detected in CIC as well as in mesangial deposits from patientswith IgAN (46). The presence of this Gal-deficient termini inthe hinge region of IgA1 could increase the exposure of antigenicdeterminants, which would be recognized by naturally occurringantibodies with anti-glycan specificity, inducing the formationof CIC (8). In the present work, we demonstrated the role ofaberrant glycosylation in favoring the binding of Gal-deficientIgA and IgA–CIC to TfR on mesangial cells. Our data showedthat IgA purified from sera of patients with IgAN has an increasedability to bind TfR expressed on HMC and on the Daudi cell line(that expresses TfR as the only IgA receptor) (25). Togetherwith our present observation that there is no TfR interactionwith a recombinant dimeric IgA1 containing the IgA2 hinge region,these data reinforce and explain our previous observation thatIgA1 but not IgA2 can bind to TfR ((25) and data presented here).Our data indicate that the binding site of IgA1 on TfR involvesthe IgA1 hinge region. However, IgA1 hinge region alone is notsufficient to promote binding to TfR as evidenced with an IgA2mutant containing IgA1 hinge region. In addition, other IgAdomains could play a role because IgA1 without N-linked carbohydratesfailed to bind the TfR. Taken together, one can conclude thatIgA1 glycosylation influences IgA1 interaction with the TfR.By analogy with other Ig, one could speculate that sugars maybe of importance for a correct folding of IgA1 (47). In addition,we still cannot exclude that some of these sugars may actuallyparticipate in the binding site itself, whereas other sugarsmay influence the binding indirectly by altering the IgA conformation.However, our experiments with deglycosylated IgA1 strongly pointto a major role of O-linked rather than N-linked sugars in theinteraction with the TfR. It is interesting that desialylationand degalactosylation of IgA1 induces a two- to threefold increasein binding to TfR as observed in Daudi cells and because bindingof these IgA1 to HMC was strongly inhibited by the sTfR1. Thismay provide a link to TfR for previous observations that IgA1molecules containing Gal-deficient O-linked glycans and CICcontaining aberrantly glycosylated IgA have an enhanced bindingto HMC (15), yet in IgAN, that IgA1 is found deposited in themesangium and that IgA1 demonstrate an altered glycosylationare consistent with our observation that IgA1 binding to theTfR (the only IgA receptor identified so far on HMC) is modulatedby the glycosylation state of IgA1. Indeed, IgA1 eluted fromkidney are hypogalactosylated (46, 48), providing further evidencethat hypogalactosylation of IgA1 promotes IgA1 mesangial depositionthrough the TfR.
Together, these data are consistent with the hypothesis on therole of aberrant IgA1 glycosylation in IgAN (49) and may beexplained by the influence of aberrant glycosylation on TfR/IgA1interaction. Furthermore, this is the first piece of evidenceshowing that altered glycosylation of the hinge region of IgA1in patients with IgAN has a direct relationship with the increasedinteraction of IgA1 with the mesangial TfR. Whether alteredglycosylation of IgA1 is the initial event resulting in increasedTfR expression in the mesangium of patients with IgAN remainsto be addressed. Finally, the absence of increase in solubleTfR levels in serum from patients suggests that increased IgA1binding to HMC in IgAN does not induce a shedding process withthe release of soluble TfR in the serum.
In conclusion, we identified for the first time that two factorsinfluence IgA1 interaction with the TfR, namely their molecularsize and glycosylation. The inhibition of IgA1 binding by solubleTfR molecules could open avenues as a complementary approachin future therapeutic strategies to be used in IgAN.
Acknowledgments
We thank Pamela J. Bjorkman (CalTech, CA) and Jean-Marie Fourneau(Necker Hospital, Paris, France) for providing the lytic baculovirus/insectcell recombinant soluble version of human TfR and recombinanthuman HLA-DR4 protein; and J. Mestecky M. Tomana, Max D. Cooper(University of Alabama at Birmingham) and N. Charles for criticalreading of the manuscript or fruitful suggestions. I.C.M. wasthe recipient of a Fondation pour la Recherche Médicale(France) and Fundação Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior(Capes-Brasil) fellowship. R.C.M. was supported by grants fromInserm and la Ligue contre le Cancer, and J.N. was supportedby grants from the National Institutes of Health (DK57750 andDK61525).
Footnotes
I.C.M and M.A.F. contributed equally to this work.
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Received for publication October 1, 2003.
Accepted for publication December 6, 2003.
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Top
Abstract
Introduction
-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR–IgA1 interaction as initial steps in IgAN pathogenesis. 
RI (10) that induces the release of a soluble form of Fc
2b
) (25), MA712 anti-TfR (
) anti-IgA1, and 14-3-26 (
6.4 and dissociates after recycling to the cell surface at pH 7.4 (37). Under physiologic conditions, no internalization of TfR is observed. However, this can be achieved by anti-TfR antibodies. Functional IgA receptors such as CD89 and the ASGP-R are internalized and degraded after binding of IgA-containing immune complexes (23). Therefore, we examined whether pIgA1 binding to TfR could mediate TfR endocytosis. 
3 in the binding of human IgE to its high-affinity receptor Fc
