Differential Segmental Flexibility and Reach Dictate the Antigen Binding Mode of Chimeric IgD and IgM: Implications for the Function of the B Cell Receptor 1 Geir Å. Løset, 2 * Kenneth H. Roux, † Ping Zhu, † Terje E. Michaelsen, ‡ and Inger Sandlie* Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H 2 L 2 Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties. The Journal of Immunology, 2004, 172: 2925–2934. T he initiation of the humoral immune response involves specific recognition of Ag by the B cell receptor (BCR) 3 . The BCR is comprised of a membrane-bound Ab unit associated with the Ig-/ heterodimer (1–3). IgD is regarded as the major BCR, coexpressed with IgM on the surface of peripheral mature, naive B cells both in human and mouse (4 – 6). However, the specific biological role of this BCR coexpression remains elu- sive despite continuing experimental efforts. In the periphery, both IgM and IgD binding to Ag can mediate B cell activation and deletion, though putative discrepancies be- tween thymus-independent and thymus-dependent Ags have been observed (7–10). Furthermore, both Igs use essentially the same intracellular signal transduction machinery, but the signals trans- mitted differ by partly yet unknown mechanisms (11–14). However, when analyzed in a knockout mouse context both IgD and IgM are to a large extent interchangeable, which points toward a redundancy between IgM and IgD from fetal to adult immune competency (15–17). In contrast, there seems to be a selective advantage for the functional  allele in heterozygous IgD +/- knockout mice, pointing toward maintenance mechanisms of a dual IgM and IgD function (15). This report also suggests that the IgD-BCR has a role in recruitment of B cells into germinal centers, as the IgD -/- mice exhibited delayed Ab affinity maturation. A recent report further underscores this importance of the IgD-BCR in germi- nal center development in comparison to the IgM-BCR (14). Both human and murine IgD have unusually long and Cys-free hinge regions, which may influence the segmental flexibility of their Ag binding sites (18, 19). This has led to the hypothesis that IgD may be more efficient in cross-linking polyvalent Ags than IgM. Indeed, results obtained from cellular assays may indicate that such differences exist (20). However, when further dissecting the putative increased epitope sensitivity observed with the IgD- BCR, it seems only to be a consequence of the higher IgD-BCR expression level on the cells compared with the IgM-BCR (21). Thus, taken together, the current body of evidence still falls short in clarifying whether or not the IgD and IgM coexpression reflects true genetic redundancy (22). Furthermore, the vast ma- jority of the reports rely on a murine context. In contrast to the other Ab classes, there is poor conservation in the IgD molecule as murine and human IgD have very different primary and probably quaternary structures (19, 23). IgD-like molecules have also been identified in species as evolutionary distant as teleosts, but it seems that there has been a lack of preserving selective pressure, the hallmark of genetic redundancy, when entering the mammalian lineage (24 –30). In this study, we have measured the Ag binding ability of a matched set of soluble chimeric IgM and IgD with specificity for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP). All factors other than the differences in the Fab to Fc tether and the Fc itself are thus equalized. This allowed us to analyze functional differ- ences with respect to Ag binding for different epitope densities. Notably, the IgM molecules studied here are H 2 L 2 monomers and not the pentamers normally found in the circulation. We have de- termined both the Fab-Fab flexibility and the intrinsic and func- tional affinity of these molecules at high and low epitope density. We also compared IgM and IgD with a hinge truncated IgD and chimeric IgG3. For the first time, we here report differences in Ag binding between IgD and IgM harboring identical monovalent spec- ificity. The results show that differences in segmental flexibility and Fab arm reach lead to an epitope density-dependent difference in the *Department of Molecular Biosciences, University of Oslo, Oslo, Norway; † Depart- ment of Biological Science and Structural Biology Program, Florida State University, Tallahassee, FL 30306; and ‡ Division of Infectious Disease Control, Norwegian In- stitute of Public Health, Oslo, Norway Received for publication July 14, 2003. Accepted for publication December 8, 2003. 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 This work was supported by grants from the Norwegian Cancer Society and the Norwegian Research Council. 2 Address correspondence and reprint requests to Geir Å. Løset, Department of Mo- lecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo, Norway. E-mail address: g.a.loset@bio.uio.no 3 Abbreviations used in this paper: BCR, B cell receptor; NIP, 5-iodo-4-hydroxy-3- nitrophenacetyl; MS, mass spectroscopy; RU, resonance unit; UH, upper hinge; wt, wild type. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00