Immunoglobulin D (IgD) of Atlantic cod has a unique structure.

A new immunoglobulin heavy-chain gene with some homology to mammalian IgD was recently cloned from the channel catfish and Atlantic salmon, two species of teleost fish. We have cloned and sequenced a new H-chain gene from Atlantic cod (Gadus morhua L.) which has clear similarities to these genes, but which also differs in several ways. The similarities of catfish, salmon, and cod delta to the mammalian delta genes are sequence homology, location immediately downstream of IgM (mu), and expression by alternative splicing rather than class switching. A unique feature of catfish, salmon, and cod delta is the chimeric nature of the gene product, as the mu1 exon is spliced to the delta1 exon. Several unique features of cod IgD were found: (1) a deletion of the delta3, delta4, delta5, and delta6 domains described in catfish and salmon IgD, (2) a tandem duplication of a part of the delta locus including the delta1 and delta2 domains, (3) the presence of a truncated delta7 domain downstream of the deltaTM exons, and (4) the separation of the duplicated domains by a short exon (deltay) which has homology to a conserved part of the transmembrane exon 1 (TM1) of some H-chain isotypes. This unique organization of the delta locus of cod probably developed after the evolutionary split from the catfish and salmon branches.

Roles of calnexin and Ig-alpha beta interactions with membrane Igs in the surface expression of the B cell antigen receptor of the IgM and IgD classes.

Membrane Igs (mIgs) of all five classes are associated with Ig-alpha beta dimers on the B cell surface. While mIgM requires the presence of these two associated molecules for its surface expression, mIgD does not. To study the structural basis for this differential Ig-alpha beta dependence, we created mutant mIgM and mIgD molecules (chimeras and those with reciprocal point mutations in their transmembrane sequences) and identified two amino acid residues in the transmembrane region of the mIg heavy chains responsible for this transport difference. Without Ig-alpha beta, mIgM and mutant mIgD molecules remained endoglycosidase H sensitive, consistent with endoplasmic reticulum (ER) localization. The molecular chaperone calnexin has previously been implicated in retaining unassembled mIgs in the ER. However, we found that inhibition of the association between calnexin and newly synthesized mIgs by castanospermine treatment did not lead to surface expression of normally retained mIgs. Therefore, calnexin cannot be the only retention molecule. Furthermore, for both wildtype and mutant mIgDs, association with calnexin was rather transient, thereby ruling out calnexin being a major ER retention molecule for mIgDs. Our study with castanospermine also showed that calnexin is required for wildtype mIgD surface expression only if Ig-alpha beta is absent, while the latter alone can function to promote mIg folding, assembly, and transport. Further study using our system will help to identify novel molecules and characterize their involvement in the control of mIg transport.

Membrane immunoglobulin is characterized by distinct structural subpopulations.

Membrane immunoglobulins are integral proteins on B cell surfaces that bind foreign antigens and are critically involved in the regulation of the immune response. Based upon the model of serum IgG, it has been assumed that membrane immunoglobulins are essentially four chain disulfide-linked structures of the form H2L2, where H represents an immunoglobulin heavy chain, and L a light chain. We show here that membrane immunoglobulins of the mu and delta isotypes are present on spleen cell surfaces in a much more diverse group of disulfide linked structures. In some cases mIg is linked into structures as large as H5L5, while in other instances mu or delta chains appear to be linked by disulfide interactions to non-immunoglobulin molecules. These various structural complexes may represent distinct functional entities, as the association of mIg with the cytoskeleton after mIg cross-linking appears to depend upon its structural subgroup.

The immunoaugmenting properties of murine IgD reside in its C delta 1 and C delta 3 regions: potential role for IgD-associated glycans.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.