Human IgD Research Articles

Crystal structures of the human IgD Fab reveal insights into CH1 domain diversity

Antibodies of the IgD isotype remain the least well characterized of the mammalian immunoglobulin isotypes. Here we report three-dimensional structures for the Fab region of IgD, based on four different crystal structures, at resolutions of 1.45–2.75 Å. These IgD Fab crystals provide the first high-resolution views of the unique Cδ1 domain. Structural comparisons identify regions of conformational diversity within the Cδ1 domain, as well as among the homologous domains of Cα1, Cγ1 and Cμ1. The IgD Fab structure also possesses a unique conformation of the upper hinge region, which may contribute to the overall disposition of the very long linker sequence between the Fab and Fc regions found in human IgD. Structural similarities observed between IgD and IgG, and differences with IgA and IgM, are consistent with predicted evolutionary relationships for the mammalian antibody isotypes.

Phenotyping complex human B cell compartments using high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses.

Abstract The human B cell compartment is very complex, consisting of numerous B cell subsets. Classifying this heterogeneous population into discrete subsets based on surface phenotype alone remains a major challenge in the field of B cell biology. Our lab has discovered a novel mouse IgM +IgD low/−B cell population that induces Treg proliferation in a GITRL-dependent manner, making it a potentially significant therapeutic target for autoimmune diseases. But due to their rarity and lack of specific cell surface identifiers, a more definitive phenotype is required to track this B cell subset in humans. The objective of these studies is to characterize human IgD low/−B cells from peripheral blood using both high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses. Spectral flow cytometry has the major advantage over its conventional predecessor by allowing for the construction of large parameter panels that are not limited by standard compensation. Here, we have adapted a standardized gating scheme (CD27 vs. IgD and CD24 vs. CD38) that captures all the major canonical human B cell subsets. Furthermore, we have used analysis plugins in FlowJo (Phenograph and FlowSOM) that predict multiple discrete B cell subpopulations within the IgD low/−phenotype. Additionally, we have analyzed human B cells (CD19 +IgM +IgD low/−) using scRNA-seq combined with CITE-seq. This approach allowed us to analyze both the cell surface protein expression of CD27, CD21, CD24, and CD38 combined with the transcriptome at the single-cell level. Our analysis has revealed four individual subclusters within this populationthat can be validated by flow cytometry, showcasing the power of combining these two cutting-edge analytical tools.

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review.

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Biophysical evaluation of hybrid Fc fusion protein of hGH to achieve basal buffer system

A newly developed hybrid Fc (hyFc) is a non-immunogenic and non-cytolytic Fc with intact Ig structure derived from human IgD and IgG4. It is fused with the human growth hormone (GXD-9) and was evaluated by various biophysical techniques. Two thermal transitions were evident by DSC, reflecting the unfolding of IgG4 and the conjugated protein. The highest Tm of the initial GXD-9 was 68.17°C and the Tm of the two domains were around 66°C and 70°C. Although Tm increased with decreasing concentration, which reflects increasing conformational stability, aggregation issues were still observed by DLS. This might be caused by decreasing or low zeta potential due to a highly complex structure. The protein was dialyzed to various pH (6.2–8.2) values to enhance conformational stability and to overcome aggregation issues. The results of CD spectroscopy were correlated with DSC measurements to evaluate its conformational stability. Changes in secondary structural contents were similar as determined by DSC and DLS. In conclusion, GXD-9 was found to be most stable at pH 7.0. The investigation of the biophysical stability of a hyFc-fusion protein has demonstrated a positive feasibility of developing more stable formulations to facilitate the initial drug development process for further clinical trials.

HIgD promotes human Burkitt lymphoma Daudi cell proliferation by accelerated G1/S transition via IgD receptor activity.

The aim of the present study was to investigate the role and molecular mechanism of human IgD (hIgD) on the proliferation of human Burkitt lymphoma Daudi cells in vitro. Logarithmically growing Daudi cells were treated with hIgD for different time periods, and cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. The expressions of Daudi surface markers and IgD receptor (IgDR) as well as cell cycle and apoptosis were measured by flow cytometry analysis. Our results showed that hIgD stimulation induced proliferation and IgDR expression and reduced the apoptosis of Daudi cells. Treatment with hIgD promoted progression of the cell cycle at the G1/S transition, and this was accompanied by upregulation of c-myc, cyclin D3, and CDK6 as well as downregulation of p16 mRNA and protein levels. Moreover, hIgD treatment also upregulated the expression of tyrosine phosphorylation of 70 kDa protein (IgDR) and p-Lyn. Taken together, these results indicate that hIgD can induce Daudi cell proliferation through activating IgDR to initiate the tyrosine phosphorylation signaling cascade to accelerate the G1/S transition.

Controlled release of human growth hormone fused with a human hybrid Fc fragment through a nanoporous polymer membrane

Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and structural integrity of hGH. Thus, the nanoporous release device could be a potential delivery system for the long-term controlled release of therapeutic proteins fused with the hybrid Fc fragment.

Aberrant Expression of Neuronal Receptors in CLL: Potential Targets with Therapeutic Implications

Abstract 3899 Introduction:Combination chemo-immunotherapy has improved the survival in previously untreated CLL, but the treatment of relapsed and fludarabine refractory CLL continues to be challenging. Inhibiting the B-cell receptor (BCR) signalling pathway appears to be beneficial in the majority of patients but is not sufficient to eradicate disease and combination approaches are already being investigated. We have previously undertaken large scale protein expression profiling in CLL and identified several molecules involved in neurotransmission that are highly expressed in CLL. Aim:To explore signalling mechanisms in neuronal markers that are aberrantly expressed in CLL, and to determine whether they have therapeutic potential in combination with BCR inhibitors. Methods:RNA expression data (Haslinger et al; Journal of Clinical Oncology. 2004) was used to identify candidate antigens that could be screened for protein level expression. Screening was restricted to antigens expressed on plasma membrane with an extracellular epitope and a commercially available antibody suitable for testing in combination with mouse monoclonal gating antibodies (i.e. fluorochrome-conjugated monoclonal, non-conjugated mouse monoclonal labelled using the zenon technique, or rabbit polyclonal detected using fluorochrome-conjugated secondary goat F(ab')2 anti-rabbit antibody). In vitro viability experiments were performed using either mononuclear cells, separated using lymphoprep, or purified CLL cells isolated with a B-cell isolation kit (Miltenyi Biotec), cultured with or without the presence of CD40L transfected fibroblasts as a supporting stromal layer. Viability was assessed using Annexin-V, 7-AAD and counting beads.To study pathway interactions, the B-cell receptor was stimulated by crosslinking using goat F(ab')2anti-human IgM or IgD and signalling responses were assessed by Syk phosphorylation status (Syk pY348 PE, BD Phosflow) and calcium flux (Fluo-3/Fura red, Invitrogen). Results:84 target antigens were assessed of which 11 showed binding on all mononuclear cells and 9/84 showed binding on CLL cells and/or normal B-cells but not other leucocytes. Of the B-cell targets, 4/9 molecules have a role in neurotransmission, such as the nicotinic acetyl choline receptor subunit β4 (CHRNB4) and dopamine receptor D4 (DRD4). CHRNB4 had the highest level of expression on B-cell and CLL surface membranes and therefore other subunits of nicotinic acetyl choline receptor were further evaluated using RT-PCR. Of the 15 subunits tested 10 were expressed on CLL cells suggesting the possibility of an important role in the pathophysiology of CLL. However, in vitro experiments did not show a significant effect on viability of CLL cells with either acetyl choline or its antagonist mecamylamine.Unlike acetyl choline, dopamine was found to be effective in reducing CLL cell survival in vitro, in a dose dependent manner with an LC50 of 8.2μM (95%CI- 5.2 to 12.9, n=6). Assessment of dopamine receptor agonists and agonists demonstrated that the D2-like family specific antagonist domperidone enhances the activity of dopamine and also has single agent activity in reducing in vitro CLL cell viability.To determine the effect of dopamine on BCR pathway signalling, syk phosphorylation and calcium flux after IgD crosslinking were measured. Stimulation of the BCR pathway by crosslinking using goat F(ab’)2 anti human IgD increased the mean fluorescent intensity of syk phosphorylation by 1.9 fold (p=0.0024) compared to 1.5 fold (p=0.0186) when pre-incubated with dopamine and 1.2 fold (p=0.0059) with a syk inhibitor. Similarly the mean percentage rise in CLL cells fluxing calcium has fallen from 20.1% to 8.2% (p=< 0.0001) when treated with dopamine and to 5.6% (p=< 0.0001) when treated with syk inhibitor. In vitro testing demonstrated a synergistic effect in blocking of the D2 and the BCR pathways as a combination of PI3 kinase δ inhibitor, GS-1101, along with domperidone reduced CLL cell viability more efficiently than either agent alone. Conclusion:Systematic screening has identified several new targets in CLL, in particular several neuronal markers which are highly expressed and functionally active. Biological investigations suggest that these neuronal receptors could play an important role in the pathophysiology of CLL and show potential synergies with therapeutically active BCR inhibitors. Disclosures:Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Identification of Two IgD+ B Cell Populations in Channel Catfish,Ictalurus punctatus

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

Measurement of human serum IgD levels.

This unit describes an ELISA for the quantitative measurement of IgD levels in human serum. The ELISA is highly specific and sensitive, with a minimum detectable concentration of 30 pg/ml and more than 10,000-fold specificity for IgD over all other human immunoglobulins. Linear dilution characteristics enable measurement of IgD concentrations ranging over 5 orders of magnitude. These factors are vital for the IgD assay, since IgD makes up only a small proportion of the total immunoglobulins present in normal sera, and IgD serum concentrations are known to vary widely between individuals.

Selective concentration of IgD class-specific antibodies in human milk

The participation of human IgD class antibody in local immune responses of breast tissue was studied by analysing the sera-to-milk ratios of total IgD, IgM, IgA, IgG isotypes and albumin found in matched samples, and by analysing the sera-to-milk (S/M) ratios of IgD, IgM, IgA, IgG antibodies against Haemophilus influenzae capsular polysaccharide (PRP), phosphorylcholine, tetanus and in some cases diphtheria antigens. The study group consisted of eight women immunized during pregnancy with PRP, and control, unimmunized women. Albumin, and total IgG showed high S/M ratios. IgA had a low S/M ratio as expected, consistent with reports that IgA is locally concentrated. Total IgD and IgM isotype ratio values were intermediate between IgG and IgA suggesting they were selectively concentrated in breast fluids due to local production or transport mechanisms, or both. Ratios for specific antibodies of IgA and IgM isotypes and for total IgA and IgM isotype showed parallel data. Among the IgD antibodies, those specific for PRP and phosphorylcholine suggested a higher degree of selective concentration as compared with tetanus antigen. In the group of unimmunized women, although selective concentration of total IgD was observed, specific antibody studies were inconclusive due to the low milk IgD antibody levels encountered. The results indicate that IgD (and also IgM) may participate in local immune responses of human breast tissues and fluids; possibly influenced by the nature of the antigen, the state of immunization and the hormonal environment (pregnancy).

Moraxella catarrhalis-dependent tonsillar B cell activation does not lead to apoptosis but to vigorous proliferation resulting in nonspecific IgM production

The respiratory pathogen Moraxella catarrhalis has a high affinity for human IgD and is mitogenic for peripheral blood B lymphocytes. Moraxella IgD-binding protein, which is a multifunctional outer membrane protein with adhesive properties, is responsible for the interaction. Previous experiments with the Ig-binding B cell superantigens protein A and protein L from Staphylococcus aureus and Peptostreptococcus magnus, respectively, have suggested that nonimmune BCR cross-linking induces B cell apoptosis through the intrinsic pathway. The goal of this study was to characterize early and late B cell events in the presence of M. catarrhalis in comparison with S. aureus. Despite an increased phosphatidyl serine translocation as revealed by Annexin V binding in flow cytometry analyses, neither M. catarrhalis nor S. aureus induced activation-associated apoptotic cell death in purified human tonsillar B cells. In contrast, a vigorous B cell proliferation, as quantified using thymidine incorporation and CFSE staining, was observed. An increased expression of an array of surface proteins (i.e., CD19, CD21, CD40, CD45, CD54, CD69, CD86, CD95, and HLA-DR) and IgM production was found upon activation with M. catarrhalis. In conclusion, M. catarrhalis-dependent B cell activation does not result in apoptosis but in cell division and nonspecific IgM synthesis, suggesting that the bacterial interaction with tonsillar B cells serves to redirect the early adaptive immune response.

The IgD CH1 region contains the binding site for the human respiratory pathogen Moraxella catarrhalis IgD‐binding protein MID

The Moraxella catarrhalis IgD-binding protein (MID) has a unique specificity for human IgD, and the sequence with maximal IgD binding is located within the amino acids MID962-1200. In the present paper, we examined the MID binding site on IgD using a series of recombinant Ig. Full-length IgD, IgD F(ab')2, and an IgD F(ab') C290R mutant lacking the inter-heavy-chain cysteine 290 were manufactured. Furthermore, a series of IgD/IgG chimeras were constructed. ELISA, dot blot and flow cytometry were used to study the binding of purified Ig to native MID, recombinant MID962-1200 or to Moraxella with or without MID. MID962-1200 bound both the IgD F(ab')2 and F(ab') C290R, indicating that the binding occurred independently of antibody structure. When amino acids 157-224 of the IgD CH1 region were substituted with IgG sequences, binding by M. catarrhalis or recombinant MID962-1200 was abolished. Subsequent smaller substitutions of IgD CH1 157-224 with IgG sequences led us to conclude that IgD CH1 amino acids 198-206 were crucial for the interaction between MID and IgD.

VH1–46 Is the Dominant Immunoglobulin Heavy Chain Gene Segment in Rotavirus-Specific Memory B Cells Expressing the Intestinal Homing Receptor α4β7

Memory B cells expressing the intestinal homing marker alpha4beta7 are important for protective immunity against human rotavirus (RV). It is not known whether the B cell repertoire of intestinal homing B cells differs from B cells of the systemic compartment. In this study, we analyzed the RV-specific VH and VL repertoire in human IgD- B cells expressing the intestinal homing marker alpha4beta7. The mean frequency of RV-specific B cells in the systemic compartment of healthy adult subjects was 0.6% (range, 0.2-1.2). The mean frequency of IgD- B cells that were both RV specific and alpha4beta7 was 0.04% (range, 0.01-0.1), and a mean of 10% (range, 1-32) of RV-specific peripheral blood human B cells exhibited an intestinal homing phenotype. We previously demonstrated that VH1-46 is the dominant Ab H chain gene segment in RV-specific systemic B cells from adults and infants. RV-specific systemic IgD- or intestinal homing IgD-/alpha4beta7+ B cells in the current study also used the gene segment VH1-46 at a high frequency, while randomly selected B cells with those phenotypes did not. These data show that VH1-46 is the immunodominant gene segment in human RV-specific effector B cells in both the systemic compartment and in intestinal homing lymphocytes. The mean replacement/silent mutation ratio of systemic compartment IgD- B cells was >2, consistent with a memory phenotype and antigenic selection. Interestingly, RV-specific intestinal homing IgD-/alpha4beta7+ B cells using the VH1-46 gene segment were not mutated, in contrast to systemic RV-specific IgD- B cells.

The CD19 Molecule is Crucial for MID‐Dependent Activation of Tonsillar B Cells from Children

The Moraxella immunoglobulin (Ig) D-binding protein (MID) induces a strong proliferative response in human peripheral blood IgD+ B cells from adults isolated by positive selection using anti-CD19-conjugated microbeads. Here, we show that tonsillar B cells from children isolated with positive selection are unable to respond to MID stimulation. The proliferative response was very low or absent at various concentrations of MID tested and at different time points analysed, whereas the MID response of tonsillar B cells from adults isolated with positive selection was considerably higher. Tonsillar B cells from children isolated with positive selection responded to formalin-fixed preparations of Moraxella catarrhalis and Staphylococcus aureus Cowan strain I. In comparison to cells isolated with positive selection, a much higher proliferative response was recorded in tonsillar B cells from children isolated with negative selection, indicating that occupation of the CD19 molecule (i.e. positive selection) inhibited the response. Indeed, the addition of anti-CD19 monoclonal antibodies (MoAb) to MID-activated tonsillar B cells from children isolated with negative selection strongly inhibited the proliferative response. In contrast, anti-CD21 MoAb at the same concentration did only show a minor inhibition on the MID-induced response. Pre-incubation of tonsillar B cells isolated from children with anti-CD19 or anti-CD21 MoAb did not affect the binding of biotin-conjugated MID as analysed by flow cytometry. These results suggest that MID-activated tonsillar B cells from children have a strong requirement for signalling through the CD19 molecule. Future experiments will further reveal the importance of CD19 and possibly other molecules for optimal activation of tonsillar B cells isolated from both children and adults.

The Glycosylation of Human Serum IgD and IgE and the Accessibility of Identified Oligomannose Structures for Interaction with Mannan-Binding Lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.

The immunoglobulin D-binding protein MID from Moraxella catarrhalis is also an adhesin.

The Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD. MID is found in the majority of M. catarrhalis strains. In the present paper, we show that MID-expressing M. catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast, M. catarrhalis isolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity. To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule. The truncated MID proteins were expressed in Escherichia coli, purified, and used for raising polyclonal antibodies in rabbits. Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID(764-913). In addition, antibodies against full-length MID, MID(764-913), or a 30-amino-acid consensus sequence (MID(775-804)) inhibited adhesion to alveolar epithelial cells. Antibodies against UspA1, an outer membrane protein expressed in essentially all M. catarrhalis strains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells. Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine against M. catarrhalis.

The Moraxella catarrhalis immunoglobulin D-binding protein MID has conserved sequences and is regulated by a mechanism corresponding to phase variation.

The prevalence of the Moraxella catarrhalis immunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains. Eighty-four percent of the clinical Moraxella strains expressed MID-dependent IgD binding. The mid gene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3' end of the gene. When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected. Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein. Interestingly, homopolymeric [polyguanine [poly(G)]] tracts were detected at the 5' ends within the open reading frames. By flow cytometry, using human IgD and fluorescein isothiocyanate-conjugated anti-IgD polyclonal antibodies, most strains showed two peaks: one high- and one low-intensity peak. All isolates expressing high levels of MID had 1, 2, or 3 triplets of G's in their poly(G) tracts, while strains not expressing MID had 4, 7, 8, or 10 G's in their poly(G) tracts or point mutations causing a putative preterminated translation. Northern blot analysis revealed that the mid gene was regulated at the transcriptional level. Experiments with nonclumping variants of M. catarrhalis proved that bacteria lost their MID expression by removing a G in their poly(G) tracts. Moraxella strains isolated from the nasopharynx or from blood and sputum specimens expressed MID at approximately the same frequency. In addition, no variation was observed between strains of different geographical origins (Australia, Europe, Japan, or the United States). MID and the mid gene were found solely in M. catarrhalis, whereas related Neisseria and Moraxella species did not express MID. Taken together, MID appears to be a conserved protein that can be found in essentially all M. catarrhalis strains. Furthermore, MID is governed by poly(G) tracts when bacteria undergo phase variation.

The Immunoglobulin D-binding Part of the Outer Membrane Protein MID from Moraxella catarrhalis Comprises 238 Amino Acids and a Tetrameric Structure

Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID(962-1200)). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID(985-1142)) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID(962-1200) was efficiently attracted to a standard IgD serum and to purified myeloma IgD(kappa) and IgD(lambda) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID(962-1200) using transposons suggested that alpha-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID(962-1200) was a tetramer. Interestingly, tetrameric MID(962-1200) attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID(962-1200) is crucial for optimal IgD-binding capacity.

The Role of Constant Region Carbohydrate in the Assembly and Secretion of Human IgD and IgA1

Immunoglobulins are glycoproteins, containing N- linked carbohydrates in the heavy chain constant regions of all isotypes and O-linked carbohydrates in the hinge regions of human IgA1 and IgD. A previous study showed that IgD synthesized in the presence of tunicamycin and lacking the three N-linked glycans on the heavy chain was not secreted (Shin, S. U., Wei, D. F., Amin, A. R., Thorbecke, G. J., and Morrison, S. L. (1992) Hum. Antibodies 3, 65-74). The contribution of each of the carbohydrates in the Fc of IgD to assembly and secretion was now analyzed by eliminating the carbohydrate addition sequence, Asn-X-Ser/Thr, through site-directed mutagenesis. Only the carbohydrate nearest the sole disulfide bond between heavy chains, which remained high mannose and appeared to be buried within the folded molecule, was found to be essential for secretion. When IgD lacked that glycan, assembly reached only the heavy/light chain half-molecule stage, and heavy chains were held inside the endoplasmic reticulum. Using benzyl 2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADG) to inhibit complete O-linked glycosylation, we found that IgA1 and IgD with incomplete hinge carbohydrates were assembled and secreted from cells. Thus, one N-linked glycan plays a structural role in IgD and is required for proper assembly and secretion, but the O-linked carbohydrates in the hinge of IgD and IgA1 are not required for folding and export.