Patel Darshana R, Wallweber Heidi J A, Yin JianPing, Shriver Stephanie K, Marsters Scot A, Gordon Nathaniel C, Starovasnik Melissa A, Kelley Robert F
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
J Biol Chem. 2004 Apr 16;279(16):16727-35. doi: 10.1074/jbc.M312316200. Epub 2004 Feb 4.
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.
B细胞成熟抗原（BCMA）是肿瘤坏死因子受体家族成员，其生理作用尚不清楚。BCMA被认为是增殖诱导配体（APRIL）和B细胞激活因子（BAFF）的受体，这两种肿瘤坏死因子配体可与多种肿瘤坏死因子受体结合，并据报道在自身免疫性疾病和癌症中发挥作用。本文给出的结果提供了对BCMA与APRIL和BAFF结合的双视角分析。首先，我们表征了单体BCMA与其配体的结合亲和力；与APRIL的高亲和力相互作用（IC50 = 11±3 nM）相比，BAFF结合亲和力（IC50 = 8±5 μM）降低了约1000倍。其次，对BCMA进行鸟枪丙氨酸扫描以绘制APRIL或BAFF结合的关键残基图谱。除了先前描述的“DXL”基序（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）外，丙氨酸扫描结果预测了BCMA中的四个氨基酸位置（Tyr13、Ile22、Gln25和Arg27），这些位置可能赋予配体特异性。Tyr13的取代对BAFF结合是可耐受的，但对APRIL结合则不可耐受。Arg27是与APRIL高亲和力结合所必需的，而该残基的取代对与BAFF的亲和力影响最小。进一步的噬菌体展示实验表明，I22K、Q25D和R27Y的单突变在APRIL与BAFF结合亲和力上差异最大。将Q25D和R27Y取代引入BCMA产生了一种双特异性变体，因为它对APRIL和BAFF具有相当的结合亲和力，IC50分别为350和700 nM。单体BCMA的I22K突变体与BAFF的结合无法检测到（IC50 > 100 μM），但与APRIL结合的亲和力与野生型BCMA相似。基于这些结果，制备了具有单一I22K突变的BCMA-Fc融合蛋白，其与APRIL结合，IC50 = 12 nM，对BAFF无可测量的亲和力。这些结果表明APRIL是BCMA的首选配体，并表明可以通过氨基酸取代进一步修饰特异性。
