The IgD C_H1 region contains the binding site for the human respiratory pathogen Moraxella catarrhalis IgD‐binding protein MID

Abstract

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.

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

“…However, in light of the finding that many human IgM 2 /IgD + plasmablasts express IgD with hypermutated VH (35) and VL (39) regions that are predicted to be incapable of binding Ag, combined with the fact that secreted IgD made by stimulated IgM 2 /IgD + plasmablasts binds to Moraxella catarrhalis and Haemophilus influenzae type a and type b (40), it is plausible that catfish secreted IgD, which was shown to be expressed in serum (22), functions through its Fc portion. For example, the IgDbinding proteins of M. catarrhalis and H. influenzae were shown to be stimulatory for human IgM 2 /IgD + B cells and bind human IgD via the Cd1 domain (68)(69)(70)(71)(72), Furthermore, in a recent study, it was demonstrated that IgD cross-linking by the superantigen M. catarrhalis IgD-binding protein leads to IgD-mediated endocytosis and B cell proliferation, because the engulfed bacteria activate various TLRs (mainly TLR 9) (73). Therefore, it is feasible that catfish IgM 2 /IgD + B cells, or even a certain subset of IgM + / IgD + B cells, could become activated by an "unidentified" pathogen, which has developed IgD-binding properties.…”

Characterization of the IgD Binding Site of Encapsulated Haemophilus influenzae Serotype b

“…Interestingly, the identified binding region of Hib to IgD bears remarkable similarity to the binding site of M. catarrhalis protein MID (16). It could therefore be possible that the presently unknown Hib IgD binding protein could be similar in structure to MID.…”

“…M. catarrhalis binds IgD via the 200-kDa outer membrane Moraxella IgD binding protein MID and the IgD binding site is located at MID 962-1200 in the central part of the molecule (14,15). Recently, we reported that the IgD binding site of MID is within the C H 1 region of IgD (16). MID also functions as an adhesin, and the sequence responsible for the adhesive properties is just adjacent to the IgD binding site (17).…”

“…All recombinant Igs used in this study were full-length Ig molecules and have been manufactured in our laboratory (16). The IgD H chain sequences used were derived from the vector pAZ6812, encoding human IgD H chain with murine V H containing dansyl chloride (DNS) specificity (19).…”

“…This was done by coating microtiter plates (C8 Starwell MAXI; Nunc) with DNS conjugated to BSA (16) and subsequent incubation at room temperature for 2 h. The plates were blocked using PBS Tween 0.05% with 1.5% OVA for 1 h. After washing, the Igs were added in serial dilutions and incubated at room temperature for 1 h. A round of washings was followed by incubation with HRP-conjugated anti-human IgD polyclonal Abs (Bethyl Laboratories) for 1 h. The plates were developed and read at 450 nm.…”