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• W1990323281 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 μm) 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 μm), 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 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 μm) 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 μm), 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. The tumor necrosis factor receptors (TNFRs) 1The abbreviations used are: TNFR, tumor necrosis factor receptor; CRD, cysteine-rich domain; TNF, tumor necrosis factor; BCMA, B cell maturation antigen; TACI, transmembrane activator and CAML interactor; BAFF, B cell-activating factor; BR3, BLyS (BAFF) receptor 3; ECD, extracellular domain; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; NNS, (A/C/GT) (A/C/G/T) (G/C) codon. 1The abbreviations used are: TNFR, tumor necrosis factor receptor; CRD, cysteine-rich domain; TNF, tumor necrosis factor; BCMA, B cell maturation antigen; TACI, transmembrane activator and CAML interactor; BAFF, B cell-activating factor; BR3, BLyS (BAFF) receptor 3; ECD, extracellular domain; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; NNS, (A/C/GT) (A/C/G/T) (G/C) codon. are a superfamily of transmembrane receptors involved in cell communication within the immune system. TNFR family members are structurally characterized by extracellular cysteine-rich domains (CRDs) that form ligand-binding motifs. Family members can be further classified based on intracellular domains that can either stimulate apoptosis through a death domain or cell proliferation through a TNF receptor-associated factor binding motif (1Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 2Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2936) Google Scholar). Downstream signaling for this subgroup of the TNFR superfamily activates the NF-κB intracellular pathway, often via TNF receptor-associated factors, ultimately resulting in cell proliferation (1Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 2Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2936) Google Scholar, 3Hatzoglou A. Roussel J. Bourgeade M.F. Rogier E. Madry C. Inoue J. Devergne O. Tsapis A. J. Immunol. 2000; 165: 1322-1330Crossref PubMed Scopus (192) Google Scholar). The corresponding TNF ligands share a common structural motif called the TNF homology domain, in the form of a β sheet jelly roll, through which the ligands trimerize for receptor activation. TNF family members are expressed in a membrane-bound form but can undergo proteolysis to produce an active soluble trimer. Generally, members of the TNFR superfamily found on B or T cells are type I transmembrane proteins that have several CRDs (1Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). There are, however, receptors in a subgroup that are expressed by B cells, are type III transmembrane proteins, and contain a reduced number of CRDs: B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and BLyS (BAFF) receptor 3 (BR3, also called BAFF-R) (4Gross J.A. Johnston J. Mudri S. Enselman R. Dillon S.R. Madden K. Xu W. Parrish-Novak J. Foster D. Lofton-Day C. Moore M. Littau A. Grossman A. Haugen H. Foley K. Blumberg H. Harrison K. Kinds-vogel W. Clegg C.H. Nature. 2000; 404: 995-999Crossref PubMed Scopus (986) Google Scholar, 5Marsters S.A. Yan M. Pitti R.M. Haas P.E. Dixit V.M. Ashkenazi A. Curr. Biol. 2000; 10: 785-788Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 6Shu H.B. Johnson H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9156-9161Crossref PubMed Scopus (137) Google Scholar, 7Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browning J.L. Ambrose C. Science. 2001; 293: 2108-2111Crossref PubMed Scopus (761) Google Scholar, 8Yu G. Boone T. Delaney J. Hawkins N. Kelley M. Ramakrishnan M. McCabe S. Qiu W.R. Kornuc M. Xia X.Z. Guo J. Stolina M. Boyle W.J. Sarosi I. Hsu H. Senaldi G. Theill L.E. Nat. Immunol. 2000; 1: 252-256Crossref PubMed Scopus (308) Google Scholar). The extracellular domain (ECD) of TACI contains two CRDs, the BCMA ECD comprises one CRD, and the ECD of BR3 contains only a partial CRD. Together with the receptor (Fn14) for the TWEAK ligand, BCMA and BR3 are the smallest members of the TNFR superfamily. Since TACI, BCMA, and BR3 lack an intracellular death domain, it is believed that these receptors are involved in cell survival, proliferation, and/or differentiation. Specifically, recent in vitro studies of BCMA presented evidence for the BCMA intracellular region interacting with TNF receptor-associated factors, leading toward downstream activation of NF-κB, ELK-1, c-Jun, and p38 pathways (3Hatzoglou A. Roussel J. Bourgeade M.F. Rogier E. Madry C. Inoue J. Devergne O. Tsapis A. J. Immunol. 2000; 165: 1322-1330Crossref PubMed Scopus (192) Google Scholar). BCMA homozygous knockout mice, however, show no distinct phenotype, suggesting a redundant or nonessential role for this receptor (9Schiemann B. Gommerman J.L. Vora K. Cachero T.G. Shulga-Morskaya S. Dobles M. Frew E. Scott M.L. Science. 2001; 293: 2111-2114Crossref PubMed Scopus (884) Google Scholar, 10Xu S. Lam K.P. Mol. Cell. Biol. 2001; 21: 4067-4074Crossref PubMed Scopus (217) Google Scholar). In contrast, recent in vivo studies with TACI reveal its critical role in B cell homeostasis and mice harboring a knockout of the TACI gene develop a fatal, lupus-like autoimmune disease (11Seshasayee D. Valdez P. Yan M. Dixit V.M. Tumas D. Grewal I.S. Immunity. 2003; 18: 279-288Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). A mouse strain expressing a BR3 receptor having intact extracellular and transmembrane domains but a disrupted intracellular domain, and thus defective for downstream signaling, has a significantly reduced number of mature peripheral B cells (7Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browning J.L. Ambrose C. Science. 2001; 293: 2108-2111Crossref PubMed Scopus (761) Google Scholar). Thus, BR3 appears to be essential for B cell survival, TACI is important for modulating the B cell population, and the physiological role of BCMA is unclear. The TNF family member BAFF is the only known ligand for BR3. BAFF-dependent B cell proliferation appears to require BR3; however, BAFF has also been reported to bind TACI and BCMA (6Shu H.B. Johnson H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9156-9161Crossref PubMed Scopus (137) Google Scholar, 8Yu G. Boone T. Delaney J. Hawkins N. Kelley M. Ramakrishnan M. McCabe S. Qiu W.R. Kornuc M. Xia X.Z. Guo J. Stolina M. Boyle W.J. Sarosi I. Hsu H. Senaldi G. Theill L.E. Nat. Immunol. 2000; 1: 252-256Crossref PubMed Scopus (308) Google Scholar, 12Wu Y. Bressette D. Carrell J.A. Kaufman T. Feng P. Taylor K. Gan Y. Cho Y.H. Garcia A.D. Gollatz E. Dimke D. LaFleur D. Migone T.S. Nardelli B. Wei P. Ruben S.M. Ullrich S.J. Olsen H.S. Kanakaraj P. Moore P.A. Baker K.P. J. Biol. Chem. 2000; 275: 35478-35485Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). APRIL, the TNF family member most closely related to BAFF, also binds TACI and BCMA (5Marsters S.A. Yan M. Pitti R.M. Haas P.E. Dixit V.M. Ashkenazi A. Curr. Biol. 2000; 10: 785-788Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 7Thompson J.S. Bixler S.A. Qian F. Vora K. Scott M.L. Cachero T.G. Hession C. Schneider P. Sizing I.D. Mullen C. Strauch K. Zafari M. Benjamin C.D. Tschopp J. Browning J.L. Ambrose C. Science. 2001; 293: 2108-2111Crossref PubMed Scopus (761) Google Scholar, 8Yu G. Boone T. Delaney J. Hawkins N. Kelley M. Ramakrishnan M. McCabe S. Qiu W.R. Kornuc M. Xia X.Z. Guo J. Stolina M. Boyle W.J. Sarosi I. Hsu H. Senaldi G. Theill L.E. Nat. Immunol. 2000; 1: 252-256Crossref PubMed Scopus (308) Google Scholar). Despite cross-reactivity with receptors, the expression patterns of BAFF and APRIL are distinct; BAFF is expressed by macrophages, monocytes, neutrophils, dendritic cells, and radioresistant stromal cells, whereas APRIL is expressed by lymphoid cells and at elevated levels by some tumor cells (13Hahne M. Kataoka T. Schroter M. Hofmann K. Irmler M. Bodmer J.L. Schneider P. Bornand T. Holler N. French L.E. Sordat B. Rimoldi D. Tschopp J. J. Exp. Med. 1998; 188: 1185-1190Crossref PubMed Scopus (457) Google Scholar, 14Moore P.A. Belvedere O. Orr A. Pieri K. LaFleur D.W. Feng P. Soppet D. Charters M. Gentz R. Parmelee D. Li Y. Galperina O. Giri J. Roschke V. Nardelli B. Carrell J. Sosnovtseva S. Greenfield W. Ruben S.M. Olsen H.S. Fikes J. Hilbert D.M. Science. 1999; 285: 260-263Crossref PubMed Scopus (1000) Google Scholar, 15Nardelli B. Belvedere O. Roschke V. Moore P.A. Olsen H.S. Migone T.S. Sosnovtseva S. Carrell J.A. Feng P. Giri J.G. Hilbert D.M. Blood. 2001; 97: 198-204Crossref PubMed Scopus (491) Google Scholar, 16Scapini P. Nardelli B. Nadali G. Calzetti F. Pizzolo G. Montecucco C. Cassatella M.A. J. Exp. Med. 2003; 197: 297-302Crossref PubMed Scopus (267) Google Scholar, 17Litinskiy M.B. Nardelli B. Hilbert D.M. He B. Schaffer A. Casali P. Cerutti A. Nat. Immunol. 2002; 3: 822-829Crossref PubMed Scopus (1011) Google Scholar, 18Gorelik L. Gilbride K. Dobles M. Kalled S.L. Zandman D. Scott M.L. J. Exp. Med. 2003; 198: 937-945Crossref PubMed Scopus (206) Google Scholar). Tight regulation of BAFF levels appears to be critical for B cell homeostasis. 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 (4Gross J.A. Johnston J. Mudri S. Enselman R. Dillon S.R. Madden K. Xu W. Parrish-Novak J. Foster D. Lofton-Day C. Moore M. Littau A. Grossman A. Haugen H. Foley K. Blumberg H. Harrison K. Kinds-vogel W. Clegg C.H. Nature. 2000; 404: 995-999Crossref PubMed Scopus (986) Google Scholar, 9Schiemann B. Gommerman J.L. Vora K. Cachero T.G. Shulga-Morskaya S. Dobles M. Frew E. Scott M.L. Science. 2001; 293: 2111-2114Crossref PubMed Scopus (884) Google Scholar, 19Mackay F. Schneider P. Rennert P. Browning J. Annu. Rev. Immunol. 2002; 21: 231-264Crossref Scopus (764) Google Scholar, 20Khare S.D. Sarosi I. Xia X.Z. McCabe S. Miner K. Solovyev I. Hawkins N. Kelley M. Chang D. Van G. Ross L. Delaney J. Wang L. Lacey D. Boyle W.J. Hsu H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3370-3375Crossref PubMed Scopus (527) Google Scholar). 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. For example, studies by Kayagaki et al. (21Kayagaki N. Yan M. Seshasayee D. Wang H. Lee W. French D.M. Grewal I.S. Cochran A.G. Gordon N.C. Yin J. Starovasnik M.A. Dixit V.M. Immunity. 2002; 17: 515-524Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) reveal attenuation of autoimmune lupus-like disease progression in mice with BR3-Fc treatment. In light of the receptor cross-reactivity, an APRIL-specific receptor would be a useful laboratory diagnostic tool or even a therapeutic agent; however, an APRIL-specific receptor has not yet been characterized. Given that TNF ligands are trimeric and can bind three molecules of the corresponding receptor, there may be significant differences in apparent affinity between ligands binding to receptors in monovalent versus multivalent forms. Previous reports of binding affinities suggest that BAFF-BCMA has nanomolar affinity; however, these studies used a bivalent receptor-Fc fusion construct that could result in measured affinities that are enhanced by avidity (5Marsters S.A. Yan M. Pitti R.M. Haas P.E. Dixit V.M. Ashkenazi A. Curr. Biol. 2000; 10: 785-788Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 6Shu H.B. Johnson H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9156-9161Crossref PubMed Scopus (137) Google Scholar, 8Yu G. Boone T. Delaney J. Hawkins N. Kelley M. Ramakrishnan M. McCabe S. Qiu W.R. Kornuc M. Xia X.Z. Guo J. Stolina M. Boyle W.J. Sarosi I. Hsu H. Senaldi G. Theill L.E. Nat. Immunol. 2000; 1: 252-256Crossref PubMed Scopus (308) Google Scholar). In this study, we characterized the APRIL and BAFF binding affinities of the extracellular domains of monomeric BCMA, BCMA-Fc, and BR3-Fc in order to determine the role that avidity could play in ligand-binding affinity. Furthermore, we have performed an alanine scan (22Sidhu S.S. Weiss G.A. Wells J.A. J. Mol. Biol. 2000; 296: 487-495Crossref PubMed Scopus (104) Google Scholar, 23Sidhu S.S. Lowman H.B. Cunningham B.C. Wells J.A. Methods Enzymol. 2000; 328: 333-363Crossref PubMed Google Scholar) of the BCMA ECD in order to identify the residues determining specificity for binding to APRIL or BAFF. Finally, 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. Reagents were obtained from the following sources: o-phenylenediamine dihydrochloride (Sigma), Streptavidin peroxidase (Roche Applied Science), IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories), Protease Complete (Roche Applied Science), anti-M13-horse-radish peroxidase (Roche Applied Science), sulfo-N-hydroxysuccinimide-biotin (Pierce). Human BAFF was expressed and purified as previously described (24Gordon N.C. Pan B. Hymowitz S.G. Yin J. Kelley R.F. Cochran A.G. Yan M. Dixit V.M. Fairbrother W.J. Starovasnik M.A. Biochemistry. 2003; 42: 5977-5983Crossref PubMed Scopus (49) Google Scholar). Human BR3-Fc (25Pelletier M. Thompson J.S. Qian F. Bixler S.A. Gong D. Cachero T. Gilbride K. Day E. Zafari M. Benjamin C. Gorelik L. Whitty A. Kalled S.L. Ambrose C. Hsu Y.M. J. Biol. Chem. 2003; 278: 33127-33133Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was a kind gift from Y.-M. Hsu (Biogen). APRIL Expression and Purification—A PCR product coding for amino acids Lys104-Leu241 of murine APRIL was subcloned into the XbaI/NotI sites of a modified pET-32a vector with a deleted S-Tag and enterokinase site to generate an N-terminal thioredoxin fusion protein. The pET-32a-APRIL(Lys104-Leu241) was transformed into Origami (DE3) competent cells (Novagen). Overnight cultures were diluted 1:100 and grown at room temperature in LB medium with 50 μg/ml carbenicillin to an A600 of 0.8 with vigorous shaking. Isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm for induction, and cultures were grown overnight at 25 °C. One liter of frozen cell pellet was resuspended in 100 ml of buffer A (50 mm Tris-HCl, pH 7.6, 300 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 2 mm benzamidine) with 5 mm imidazole and placed on ice for 30 min. Cells were homogenized by passage through a microfluidizer and centrifuged at 15,000 rpm for 45 min. Supernatant was loaded onto a 3-ml nickel-nitrilotriacetic acid-agarose column (Qiagen), washed with 10 column volumes of Buffer A with 10 mm imidazole, and eluted with 10 column volumes of Buffer A with 300 mm imidazole. Fractions containing thioredoxin-APRIL fusion protein were pooled, concentrated, and purified by Superdex 200 size exclusion chromatography. Baculovirus Expression and Purification of BCMA ECD—DNA coding for residues 4-53 of the human BCMA ECD was amplified by PCR and subcloned into the vector pET15b (Novagen) using the NdeI and XhoI restriction sites to introduce an N-terminal His tag and a thrombin cleavage sequence preceding the BCMA coding region. This His-tagged BCMA construct was subcloned into the baculovirus transfer vector pAcGP67B (Pharmingen) using the BamHI and NotI restriction sites. The transfer vector was co-transfected with BaculoGold DNA into Sf9 cells, and recombinant virus was subsequently isolated and amplified to facilitate protein production. After 3 days of growth of virally infected Hi5 cells at 27 °C, His-tagged protein was purified from the culture medium by chromatography on Ni2+-nitrilotriacetic acid resin as described previously (26Hymowitz S.G. Filvaroff E.H. Yin J.P. Lee J. Cai L. Risser P. Maruoka M. Mao W. Foster J. Kelley R.F. Pan G. Gurney A.L. de Vos A.M. Starovasnik M.A. EMBO J. 2001; 20: 5332-5341Crossref PubMed Scopus (417) Google Scholar), followed by gel filtration on a Superdex 75 column. BCMA eluted from the Superdex 75 column as a monomer. N-terminal sequencing and mass spectrometry were used to confirm the proper identity of the purified protein and the presence of both glycosylated and nonglycosylated species in the purified protein pool, respectively. BCMA-Fc Expression and Purification—DNA encoding the ECD (residues 5-51) of hBCMA was fused to hFc to form a fusion construct in the pRK vector and expressed in Chinese hamster ovary cells as previously described (5Marsters S.A. Yan M. Pitti R.M. Haas P.E. Dixit V.M. Ashkenazi A. Curr. Biol. 2000; 10: 785-788Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Site-directed mutagenesis (Stratagene QuikChange™ method) was used to introduce the single mutant I22K in the hBCMA-hFc fusion construct. Escherichia coli 294 cells were transformed with the pRK-hBCMA-hFc plasmid for large scale production of plasmid DNA. Transient transfections of the plasmid in HEK 293 cells using FuGENE 6 (Roche Applied Science) produced secreted BCMA-Fc protein. The BCMA-Fc was purified from the HEK 293 growth medium by affinity chromatography using Protein A-Sepharose (Amersham Biosciences) resin. Phage Display of BCMA—An initial vector for phage display of the BCMA extracellular domain was prepared by PCR subcloning of the fragment encoding residues 5-50 into the phagemid sTF-g3 (27Lee G.F. Kelley R.F. J. Biol. Chem. 1998; 273: 4149-4154Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The resulting construct (BCMA1-g3) contained residues 5-50 fused at the C terminus to a tripeptide (G-S-A) linker and an amber stop codon followed by the C-terminal half of the M13 p3 coat protein. The bacterial signal sequence stII was joined to the N terminus of BCMA with an inserted Ser residue comprising the P1′ cleavage site for the signal peptidase. Expression was driven by the alkaline phosphatase promoter. Phagemid BCMA2-g3 was prepared by using site-directed mutagenesis (28Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar) to insert the peptide epitope (MADPNRFRGKDLGG) for an antibody (3C8:2F4; Genentech, Inc.) between the P1 and P1′ residues of the signal sequence cleavage site. BCMA2-g3 phagemid was used to prepare two “shotgun alanine” scanning libraries essentially as described previously (29Weiss G.A. Watanabe C.K. Zhong A. Goddard A. Sidhu S.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8950-8954Crossref PubMed Scopus (251) Google Scholar). A shotgun alanine codon will code for the wild-type residue, alanine, or one of two additional substitutions in certain cases, due to codon degeneracy, at a given position. Each of these libraries, prepared separately, contains shotgun codons at unique positions. Library 1 has shotgun codons at positions 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19; library 2 has shotgun codons at positions 22, 23, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, and 36. Each library contained at least 1 × 1011 phage/ml, allowing for complete representation of the theoretical diversity (>105-fold excess) (library 1 codes 1.1 × 106 unique sequences, and library 2 codes 5.2 × 105 unique sequences). Library Sorting and Analysis—Phage from each of the libraries described above were subjected to rounds of binding selection against either BAFF, APRIL, or anti-tag antibody (3C8:2F4; Genentech, Inc.) immobilized on 96-well Nunc Maxisorp immunoplates (30Sidhu S.S. Biomol Eng. 2001; 18: 57-63Crossref PubMed Scopus (157) Google Scholar). Bovine serum albumin-coated wells were used to determine nonspecific background binding. Phage eluted from each target were propagated in E. coli XL1-Blue in the presence of M13K07 helper phage; amplified phage were used for selection against the same target in the previous round. Phage sorting was stopped, generally at round 2 or 3, when 100-fold enrichment was obtained. Enrichment was calculated from the ratio of the phage titer eluted from the target-coated wells to the phage titer eluted from the bovine serum albumin-coated wells. Individual clones from each library and selection target were then grown in a 96-well format in 400 μl of 2YT medium supplemented with carbenicillin and KO7 helper phage. Phage ELISA assays (29Weiss G.A. Watanabe C.K. Zhong A. Goddard A. Sidhu S.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8950-8954Crossref PubMed Scopus (251) Google Scholar) were performed to detect phage-displayed variants of BCMA ECD capable of binding BAFF, APRIL, or anti-tag antibody. All clones tested that were found to be positive in their respective ELISAs were then sequenced as described previously (30Sidhu S.S. Biomol Eng. 2001; 18: 57-63Crossref PubMed Scopus (157) Google Scholar). Sequences of acceptable quality were translated and aligned. For selection of binding to BAFF, 40 and 47 sequences were analyzed for libraries 1 and 2, respectively. For selection of binding to APRIL, 44 and 46 sequences were analyzed from libraries 1 and 2, respectively. For the display selection, binding to anti-tag antibody, a minimum of 40 sequences were analyzed for each library. To quantify the effect of each mutation on ligand binding, normalized frequency ratios (F) for each amino acid position were calculated from a ratio of ligand selection to display efficiency selection, as described previously (31Skelton N.J. Koehler M.F. Zobel K. Wong W.L. Yeh S. Pisabarro M.T. Yin J.P. Lasky L.A. Sidhu S.S. J. Biol. Chem. 2003; 278: 7645-7654Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). NNS Library Construction and Sorting—The BCMA2-g3 phagemid was used to prepare a library having complete randomization (NNS degenerate codon as defined by IUB code (23Sidhu S.S. Lowman H.B. Cunningham B.C. Wells J.A. Methods Enzymol. 2000; 328: 333-363Crossref PubMed Google Scholar)) of residues Leu18, Ile22, Gln25, and Arg27. The library contained 1 × 1010 phage/ml, allowing complete representation of the library, theoretically 1 × 106 unique members. This library was sorted as described for the shotgun alanine libraries. Since each amino acid position selected for NNS codon introduction has the potential of all 20 amino acids from 31 triplet codons, the data are weighed according to codon degeneracy by calculating the ratio of percentage of occurrence to percentage of degeneracy of the amino acid at a given position as suggested previously (32LaBean T.H. Kauffman S.A. Protein Sci. 1993; 2: 1249-1254Crossref PubMed Scopus (35) Google Scholar). The normalized F′ value is the percentage of occurrence to percentage of degeneracy ratio for ligand selection divided by the percentage of occurrence to percentage of degeneracy ratio for display efficiency. Expression and Purification of BCMA-Z Fusion Proteins—Plasmid BCMA-Z, designed to express a protein fusion of BCMA ECD (residues 5-50) and the Z domain of protein A (33Nilsson B. Moks T. Jansson B. Abrahmsen L. Elmblad A. Holmgren E. Henrichson C. Jones T.A. Uhlen M. Protein Eng. 1987; 1: 107-113Crossref PubMed Scopus (630) Google Scholar) was constructed by using PCR to replace the amber stop and gene 3 portion of BCMA1-g3 with the Z domain fragment from plasmid pZCT (34Starovasnik M.A. O'Connell M.P. Fairbrother W.J. Kelley R.F. Protein Sci. 1999; 8: 1423-1431Crossref PubMed Scopus (75) Google Scholar). Oligonucleotide-directed mutagenesis was performed as described (28Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar) to generate point mutations and all constructs were verified by DNA sequencing. BCMA-Z fusion proteins were expressed by secretion from E. coli and purified by chromatography on IgG-Sepharose as described previously (35Starovasnik M.A. Skelton N.J. O'Connell M.P. Kelley R.F. Reilly D. Fairbrother W.J. Biochemistry. 1996; 35: 15558-15569Crossref PubMed Scopus (50) Google Scholar). BCMA-Z proteins were further purified by size exclusion chromatography on a HiPrep 16/60 Sephacryl S-100 HR column. BCMA-Z had an elution volume from the S-100 column between that of soluble human tissue factor (Mr 24,800) and E. coli thioredoxin (Mr 11,675). A molar mass of 12,000, consistent with the monomer MW of 13017 calculated from the amino acid sequence, was calculated from light scattering data collected on a mini-DAWN detector (Wyatt Technologies). Amino acid analysis was performed on the purified BCMA-Z to determine the extinction coefficient (ϵ280 = 9832 m-1 cm-1). Competitive Displacement ELISA—Receptors were tested for binding to either APRIL or BAFF in a competition ELISA assay. A 100-μl solution of carbonate buffer (pH 9.6) containing 2 μg/ml target ligand, either APRIL or BAFF, was coated on Nunc Maxisorp 96-well plates overnight at 4 °C. The plate was washed with PBS and blocked for 1 h with 200 μl of 0.2% bovine serum albumin in PBS. In one set of experiments, 0.2 μg/ml of BCMA-Z was ad" @default.
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