Formation of Virus-like Clusters Is an Intrinsic Property of the Tumor Necrosis Factor Family Member BAFF (B Cell Activating Factor)

Abstract

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts

“…29,33 It is not known whether the activity of BAFF is conditioned by its level of oligomerization, such as those found in BAFF 3-mer and BAFF 60-mer. 13,34 To monitor the activity of BAFF 3-mer and oligomers, a surrogate cell death assay was developed, in which the oligomerization-dependent apoptotic Fas pathway can be initiated by BAFF. For this purpose, a fusion protein consisting of the extracellular domain of BCMA fused to the transmembrane and intracellular domains of Fas was expressed in Jurkat T cells.…”

“…Myc-BAFF, BAFF H218A, BAFF A134 and BAFF N242Q, Flag-BAFF, BCMA-Fc, TACI-Fc and BAFF-R-Fc have been described previously. 13,28 Flag-tagged APRIL containing amino acid 92-233 of human APRIL was purified from supernatants of transiently transfected 293T cells (Document S1). The hamster mAb B9C11 anti-mouse BAFF-R was kindly provided by Dr Melissa Starovasnik (Genentech, San Francisco, CA).…”

“…The extended DE loop of BAFF, also known as the flap region, 12 is not found in other TNF family members, but its length is precisely conserved in BAFF of fishes, batrachians, birds, and mammals ( Figure 5A). Moreover, all residues involved in trimer-trimer interaction, including the histidine residue responsible for pH-dependent assembly of the 60-mer, 12,13 are conserved in human, mouse, opossum (marsupial), platypus (monotreme), chicken, and 10 additional bird and mammalian BAFFs ( Figure 5A and data not shown).…”

“…Cell supernatants or mouse plasma (200 L) were fractionated by gel filtration on Superdex-200 as previously described 13 and analyzed by Western blotting using Buffy-2 mAb, or by ELISA (Document S1). Fractions were also tested for their ability to kill Jurkat BCMA:Fas cells.…”

“…11,12 Although the physiologic relevance of BAFF 60-mer has not been studied, recent work showed that BAFF 60-mer was also produced by cells expressing BAFF endogenously and that the 60-mer was moderately more potent than the 3-mer at costimulating BCR-induced thymidine uptake in primary B cells. 13 APRIL, a close homolog of BAFF, can also costimulate B cells but requires oligomerization to do so. 14 BAFF and APRIL share 2 receptors, namely, TACI (transmembrane activator and CAML interactor) and BCMA (B-cell maturation antigen).…”