Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor).
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.
Cachero
Teresa G
TG
Biogen Idec, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, USA. tcachero@altus.com
Schwartz
Ian M
IM
Qian
Fang
F
Day
Eric S
ES
Bossen
Claudia
C
Ingold
Karine
K
Tardivel
Aubry
A
Krushinskie
Dennis
D
Eldredge
John
J
Silvian
Laura
L
Lugovskoy
Alexey
A
Farrington
Graham K
GK
Strauch
Kathy
K
Schneider
Pascal
P
Whitty
Adrian
A
D000818:Animals; D053264:B-Cell Activating Factor; D002850:Chromatography, Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008970:Molecular Weight; D010843:Pichia / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D053264:B-Cell Activating Factor; D008565:Membrane Proteins; C505719:TNFSF13B protein, human; C505720:Tnfsf13b protein, mouse; D014409:Tumor Necrosis Factor-alpha