Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor)

TG Cachero, IM Schwartz, F Qian, ES Day… - Biochemistry, 2006 - ACS Publications
TG Cachero, IM Schwartz, F Qian, ES Day, C Bossen, K Ingold, A Tardivel, D Krushinskie…
Biochemistry, 2006ACS Publications
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≥ 7.0. More …
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 ≥ 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.
ACS Publications