Recombinant soluble trimeric CD40 ligand is biologically active.
CD40 ligand (CD40L) is expressed on the surface of activated CD4+ T cells, basophils, and mast cells. Binding of C40L to its receptor, CD40, on the surface of B cells stimulates B cell proliferation, adhesion and differentiation. A preparation of soluble, recombinant CD40L (Tyr-45 to Leu-261), containing the full-length 29-kDa protein and two smaller fragments of 18 and 14 kDa, has been shown to induce differentiation of B cells derived either from normal donors or from patients with X-linked hyper-IgM syndrome (Durandy, A., Schiff, C., Bonnefoy, J.-Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993) Eur. J. Immunol. 23, 2294-2299). We have now purified each of these fragments to homogeneity and show that only the 18-kDa fragment (identified as Glu-108 to Leu-261) is biologically active. When expressed in recombinant form, the 18-kDa protein exhibited full activity in B cell proliferation and differentiation assays, was able to rescue of B cells from apoptosis, and bound soluble CD40. Sucrose gradient sedimentation shows that the 18-kDa protein sediments as an apparent homotrimer, a result consistent with the proposed trimeric structure of CD40L. This demonstrates that a soluble CD40L can stimulate CD40 in a manner indistinguishable from the membrane-bound form of the protein.
Mazzei
G J
GJ
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Edgerton
M D
MD
Losberger
C
C
Lecoanet-Henchoz
S
S
Graber
P
P
Durandy
A
A
Gauchat
J F
JF
Bernard
A
A
Allet
B
B
Bonnefoy
J Y
JY
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D015703:Antigens, CD / Q000378:metabolism*; D017209:Apoptosis; D001402:B-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand; D002460:Cell Line; D018698:Glutamic Acid; D000882:Haplorhini; D006801:Humans; D007075:Immunoglobulin M / Q000096:biosynthesis; D007153:Immunologic Deficiency Syndromes / Q000276:immunology; D015847:Interleukin-4 / Q000494:pharmacology; D007668:Kidney; D007930:Leucine; D008024:Ligands; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D014066:Palatine Tonsil / Q000276:immunology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012016:Reference Values; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D015703:Antigens, CD; D007075:Immunoglobulin M; D008024:Ligands; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D023201:CD40 Ligand; D015847:Interleukin-4; D018698:Glutamic Acid; D007930:Leucine