Abstract
B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.
Highlights
The tumor necrosis factor receptors (TNFRs)1 are a superfamily of transmembrane receptors involved in cell communi
B cell maturation antigen (BCMA)-Z was readily purified from E. coli extracts by using a two-column procedure, and the purified protein eluted as a monomer from a gel filtration column
The competition ELISA method, as described under “Experimental Procedures,” was used to measure binding of BCMA-Fc wild type and I22K fusion proteins to a proliferation-inducing ligand (APRIL) or BAFF using biotinylated BCMA-Z for APRIL binding or biotinylated BR3 [24] for BAFF binding (Fig. 4). These data reveal that the single point mutation, I22K, in BCMA resulted in a bivalent protein that binds APRIL (IC50 ϭ 12 nM) with similar affinity to the wild type BCMA-Fc (IC50 ϭ 9 nM) but has no measurable affinity to BAFF (IC50 Ͼ 10 M)
Summary
The TNF family member BAFF is the only known ligand for BR3. BAFF-dependent B cell proliferation appears to require BR3; BAFF has been reported to bind TACI and BCMA [6, 8, 12]. BAFF knockout mice display significant reduction in the development and survival of follicular and marginal B cells, whereas mice expressing a BAFF transgene develop a lupus-like autoimmune syndrome [4, 9, 19, 20]. Based on their roles in B cell-mediated immunity, these ligands (BAFF and APRIL) and their corresponding three receptors (BR3, BCMA, and TACI) provide likely targets for disease therapy. Based on the alanine scan results, we produced a BCMA-Fc fusion with a single point mutation in the BCMA ECD, I22K, that maintains high affinity for APRIL but has no measurable binding to BAFF
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