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.
Biogen Idec, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, USA. tcachero@altus.com
Teresa G
TG
Cachero
Ian M
IM
Schwartz
Fang
F
Qian
Eric S
ES
Day
Claudia
C
Bossen
Karine
K
Ingold
Aubry
A
Tardivel
Dennis
D
Krushinskie
John
J
Eldredge
Laura
L
Silvian
Alexey
A
Lugovskoy
Graham K
GK
Farrington
Kathy
K
Strauch
Pascal
P
Schneider
Adrian
A
Whitty
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